Methods of treating bladder cancer

ABSTRACT

The present invention provides methods and compositions for treating bladder cancer, including metastatic bladder cancer and non-muscle-invasive bladder cancer, by administering a composition comprising nanoparticles that comprise mTOR inhibitor and optionally an albumin.

RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional ApplicationNo. 61/786,167, entitled “Methods of Treating Bladder Cancer,” filedMar. 14, 2013 and U.S. Provisional Application No. 61/786,175, entitled“Methods of Treating Bladder Cancer,” filed Mar. 14, 2013, the contentsof each of which are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to methods and compositions for thetreatment of bladder cancer by administering compositions comprisingnanoparticles that comprise a limus drug and an albumin.

BACKGROUND

In the US, bladder cancer is the fourth most common type of cancer inmen and the ninth most common cancer in women. Smoking and age are themain known risk factors, with nearly 90% of patients over age of 55. In2011, it is estimated that there will be 69,250 new cases in the US,resulting in 14,990 deaths. Non-muscle invasive bladder cancer (NMIBC)begins and stays in the cells lining the bladder without growing intothe deeper main muscle layer of the bladder, and accounts for themajority (70-80%) of patients diagnosed with bladder cancer (stages Ta,T1, or CIS). Approximately 30% of patients present with muscle-invasivedisease (stages T2-T4). Bladder cancer has the highest recurrence rateof any malignancy. Although NMIBC is a relatively benign disease, itrecurs in 50-70% of patients, of which 10-20% would eventually progressto high-grade muscle-invasive disease. Furthermore, the disease is alsocharacterized by having a large pool of patients who have beenpreviously diagnosed and are still undergoing treatment for unresolvedtumors; more than 1 million patients in the US and Europe are estimatedto be affected by the disease.

The high-grade muscle invasive disease is typically treated with radicalcystectomy or a combination of radiation therapy and chemotherapy.However, even after treatment the tumor usually remains and patients areat risk of tumor progression, leading to a shortened life expectancy ordeath from metastatic disease. Approximately 350,000 patients in the USand EU are currently undergoing treatment for unresolved tumors.

NMIBC is typically treated with intravesicular BCG, which elicits anonspecific local immune response against the tumor cells. BCG elicits anonspecific massive local inflammatory reaction in the bladder wall, andelevated appearance of cytokines can be detected in the urine ofBCG-treated patients. BCG is internalized by antigen-presenting cells,such as macrophages, but also by urothelial tumor cells, which result inan altered gene expression of these cells. Additionally, none of theavailable chemotherapeutic agents, including gemcitabine, cisplatin, andvalrubicin, that are currently explored in clinical trials for treatingbladder cancer are targeted therapeutics.

Sirolimus (INN/USAN), also known as rapamycin, is an immunosuppressantdrug used to prevent rejection in organ transplantation; it isespecially useful in kidney transplants. It prevents activation of Tcells and B cells by inhibiting their response to interleukin-2 (IL-2).The mode of action of sirolimus is to bind the cytosolic proteinFK-binding protein 12 (FKBP12), and the sirolimus-FKBP12 complex in turninhibits the mammalian target of sirolimus (mTOR) pathway by directlybinding the mTOR Complex1 (mTORC1).

Albumin-based nanoparticle compositions have been developed as a drugdelivery system for delivering substantially water insoluble drugs. See,for example, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868, and6,537,579, 7,820,788, and 7,923,536. Abraxane®, an albumin stabilizednanoparticle formulation of paclitaxel, was approved in the UnitedStates in 2005 and subsequently in various other countries for treatingmetastatic breast cancer. It was recently approved for treatingnon-small cell lung cancer in the United States, and has also showntherapeutic efficacy in various clinical trials for treatingdifficult-to-treat cancers such as bladder cancer and melanoma. Albuminderived from human blood has been used for the manufacture of Abraxane®as well as various other albumin-based nanoparticle compositions.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of treating bladder cancer in anindividual, comprising administering to the individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor (such as a limus drug). In some embodiments, there is provideda method of treating bladder cancer in an individual, comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the limus drug is sirolimus. In some embodiments, thealbumin is human albumin (such as human serum albumin). In someembodiments, the nanoparticles comprise sirolimus coated with albumin.In some embodiments, the average particle size of the nanoparticles inthe nanoparticle composition is no more than about 200 nm (such as nogreater than about 150 nm). In some embodiments, the compositioncomprises the albumin stabilized nanoparticle formulation of sirolimus(Nab-sirolimus). In some embodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of treating bladdercancer in an individual, comprising administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug is coated with thealbumin. In some embodiments, there is provided a method of treatingbladder cancer in an individual, comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the average particlesize of the nanoparticles in the nanoparticle composition is no greaterthan about 200 nm (such as less than about 150 nm). In some embodiments,there is provided a method of treating bladder cancer in an individual,comprising administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug is coated with the albumin, and whereinthe average particle size of the nanoparticles in the nanoparticlecomposition is no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, there is provided a method oftreating bladder cancer in an individual, comprising administering tothe individual an effective amount of a composition comprisingNab-sirolimus. In some embodiments, there is provided a method oftreating bladder cancer in an individual, comprising administering tothe individual an effective amount of Nab-sirolimus.

In some embodiments, the composition is administered intravenously. Insome embodiments, the composition is administered intravesicularly (forexample via urethral catheterization).

Also provided are combination therapy methods for treating bladdercancer. For example, in some embodiments, there is provided a method oftreating bladder cancer in an individual, comprising administering tothe individual (a) an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a limus drug); and(b) an effective amount of another agent (such as BCG). In someembodiments, there is provided a method of treating bladder cancer in anindividual, comprising administering to the individual (a) an effectiveamount of a composition comprising nanoparticles comprising a limus drugand albumin; and (b) an effective amount of another agent (such as BCG).The nanoparticle composition and the other agent can be administeredsimultaneously or sequentially. In some embodiments, the nanoparticlecomposition and the other agent are administered concurrently. In someembodiments, the limus drug is sirolimus. In some embodiments, thealbumin is human serum albumin. In some embodiments, the nanoparticlescomprise sirolimus coated with albumin. In some embodiments, the averageparticle size of the nanoparticles in the nanoparticle composition is nomore than about 200 nm (such as no greater than about 200 nm). In someembodiments, the composition comprises the albumin stabilizednanoparticle formulation of sirolimus (Nab-sirolimus). In someembodiments, the composition is Nab-sirolimus.

In some embodiments, the method is carried out in a neoadjuvant setting.In some embodiments, the method is carried out in an adjuvant setting.In some embodiments, the method is carried out after resection ofvisible tumor in the bladder.

Bladder cancer that can be treated with methods described hereininclude, but are not limited to, metastatic bladder cancer,non-muscle-invasive bladder cancer, or bladder cancer that is refractoryto a standard therapy (such as Bacillus Calmette-Guérin (BCG)) orrecurrent after the standard therapy. In some embodiments, the bladdercancer is BCG-refractory non-muscle-invasive bladder cancer. In someembodiments, the bladder cancer is platinum-refractory bladder cancer.In some embodiments, the bladder cancer is platinum-refractorymetastatic urothelial carcinoma. In some embodiments, the treatment isfirst line treatment. In some embodiments, the treatment is second linetreatment.

In some embodiments, there is provided a method of treating bladdercancer in an individual, comprising intravesicularly administering (forexample via urethral catheterization) to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin. In some embodiments, there is provided a method oftreating non-muscle-invasive bladder cancer in an individual, comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the individual has progressed from an earlier therapy forbladder cancer. In some embodiments, the individual is refractory to anearlier therapy for bladder cancer. In some embodiments, the individualhas recurrent bladder cancer. In some embodiments, there is provided amethod of treating a BCG-refractory non-muscle-invasive bladder cancerin an individual, comprising intravesicularly administering (for examplevia urethral catheterization) to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, the amount of the nanoparticle compositionis about 5 mg to about 500 mg, including for example about 30 mg toabout 400 mg (such as about 100 mg). In some embodiments, thenanoparticle composition is administered weekly.

Also provided are methods of treating bladder cancer according to anyone of the methods described above, wherein the treatment is based onthe level of one or more biomarkers.

The methods described herein can be used for any one or more of thefollowing purposes: alleviating one or more symptoms of bladder cancer,delaying progressing of bladder cancer, shrinking tumor size in bladdercancer patient, inhibiting bladder cancer tumor growth, prolongingoverall survival, prolonging disease-free survival, prolonging time tobladder disease progression, preventing or delaying bladder cancermetastasis, reducing (such as eradiating) preexisting bladder cancermetastasis, reducing incidence or burden of preexisting bladder cancermetastasis, preventing recurrence of bladder cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treatingbladder cancer by administering a composition comprising nanoparticlescomprising an mTOR inhibitor (hereinafter also referred to as “mTORnanoparticle composition”). In some embodiments, the compositioncomprises a limus drug and an albumin (hereinafter also referred to as“limus nanoparticle composition”). Also provided are compositions (suchas pharmaceutical compositions), medicine, kits, and unit dosages usefulfor the methods described herein.

Definitions

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the recurrence of thedisease, reducing recurrence rate of the disease, delay or slowing theprogression of the disease, ameliorating the disease state, providing aremission (partial or total) of the disease, decreasing the dose of oneor more other medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. Also encompassed by “treatment” is a reduction ofpathological consequence of bladder cancer. The methods of the inventioncontemplate any one or more of these aspects of treatment.

The term “individual” refers to a mammal and includes, but is notlimited to, human, bovine, horse, feline, canine, rodent, or primate.

As used herein, an “at risk” individual is an individual who is at riskof developing bladder cancer. An individual “at risk” may or may nothave detectable disease, and may or may not have displayed detectabledisease prior to the treatment methods described herein. “At risk”denotes that an individual has one or more so-called risk factors, whichare measurable parameters that correlate with development of bladdercancer, which are described herein. An individual having one or more ofthese risk factors has a higher probability of developing cancer than anindividual without these risk factor(s).

“Adjuvant setting” refers to a clinical setting in which an individualhas had a history of bladder cancer, and generally (but not necessarily)been responsive to therapy, which includes, but is not limited to,surgery (e.g., surgery resection), radiotherapy, and chemotherapy.However, because of their history of bladder cancer, these individualsare considered at risk of development of the disease. Treatment oradministration in the “adjuvant setting” refers to a subsequent mode oftreatment. The degree of risk (e.g., when an individual in the adjuvantsetting is considered as “high risk” or “low risk”) depends upon severalfactors, most usually the extent of disease when first treated.

“Neoadjuvant setting” refers to a clinical setting in which the methodis carried out before the primary/definitive therapy.

As used herein, “delaying” the development of bladder cancer means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease. This delay can be of varying lengths of time, depending onthe history of the disease and/or individual being treated. As isevident to one skilled in the art, a sufficient or significant delaycan, in effect, encompass prevention, in that the individual does notdevelop the disease. A method that “delays” development of bladdercancer is a method that reduces probability of disease development in agiven time frame and/or reduces the extent of the disease in a giventime frame, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a statistically significantnumber of subjects. Bladder cancer development can be detectable usingstandard methods, including, but not limited to, computerized axialtomography (CAT scan), Magentic Resonance Imaging (MRI), ultrasound,clotting tests, arteriography, biopsy, urine cytology, and cystoscopy.Development may also refer to bladder cancer progression that may beinitially undetectable and includes occurrence, recurrence, and onset.

As used herein, by “combination therapy” is meant that a first agent beadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality, such as administration of a nanoparticlecomposition described herein in addition to administration of the otheragent to the same individual. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to bladder cancer, aneffective amount comprises an amount sufficient to cause a tumor toshrink and/or to decrease the growth rate of the tumor (such as tosuppress tumor growth) or to prevent or delay other unwanted cellproliferation in bladder cancer. In some embodiments, an effectiveamount is an amount sufficient to delay development of bladder cancer.In some embodiments, an effective amount is an amount sufficient toprevent or delay recurrence. In some embodiments, an effective amount isan amount sufficient to reduce recurrence rate in the individual. Aneffective amount can be administered in one or more administrations. Inthe case of bladder cancer, the effective amount of the drug orcomposition may: (i) reduce the number of bladder cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent andpreferably stop bladder cancer cell infiltration into peripheral organs;(iv) inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrenceand/or recurrence of tumor; (vii) reducing recurrence rate of tumor,and/or (viii) relieve to some extent one or more of the symptomsassociated with bladder cancer.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

As used herein, the term “sequential administration” means that thefirst therapy and second therapy in a combination therapy areadministered with a time separation of more than about 15 minutes, suchas more than about any of 20, 30, 40, 50, 60, or more minutes. Eitherthe first therapy or the second therapy may be administered first. Thefirst and second therapies are contained in separate compositions, whichmay be contained in the same or different packages or kits.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Methods of Treating Bladder Cancer

The present invention provides methods of treating bladder cancer in anindividual (such as human) comprising administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (such as a limus drug). In some embodiments, theinvention provides methods of treating bladder cancer in an individual(e.g., human) comprising administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin. In some embodiments, the method comprises administeringto the individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin, wherein the limusdrug in the nanoparticles is coated with the albumin. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, the method comprises administeringto the individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin, wherein thenanoparticles comprise a limus drug coated with albumin, wherein thenanoparticles have an average particle size of no greater than about 200nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

“mTOR inhibitor” used herein refers to inhibitors of mTOR. mTOR is aserine/threonine-specific protein kinase downstream of thephosphatidylinositol 3-kinase (PBK)/Akt (protein kinase B) pathway, anda key regulator of cell survival, proliferation, stress, and metabolism.mTOR pathway dysregulation has been found in many human carcinomas, andmTOR inhibition produced substantial inhibitory effects on tumorprogression. mTOR inhibitors described herein include, but are notlimited to, BEZ235 (NVP-BEZ235), everolimus (also known as RAD001 andsold under the trademarks Zortress®, Certican®, and Afinitor®),rapamycin (also known as sirolimus and sold under the trademarkRapamune®), AZD8055, temsirolimus (also known as CCI-779 and sold underthe trademark Torisel®), PI-103, Ku-0063794, INK 128, AZD2014,NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torin 1, WAY-600,WYE-125132, WYE-687, GSK2126458, PF-05212384 (PKI-587), PP-121, OSI-027,Palomid 529, PP242, XL765, GSK1059615, WYE-354, and eforolimus (alsoknown as ridaforolimus or deforolimus).

In some embodiments, the mTOR inhibitor is a limus drug, which includessirolimus and its analogues. Examples of limus drugs include, but arenot limited to, temsirolimus (CCI-779), everolimus (RAD001),ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578),pimecrolimus, and tacrolimus (FK-506). In some embodiments, the limusdrug is selected from the group consisting of temsirolimus (CCI-779),everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669),zotarolimus (ABT-578), pimecrolimus, and tacrolimus (FK-506).

In some embodiments, the bladder cancer is a low grade bladder cancer.In some embodiments, the bladder cancer is a high grade bladder cancer.In some embodiments, the bladder cancer is invasive. In someembodiments, the bladder cancer is non-invasive. In some embodiments,the bladder cancer is non-muscle invasive.

In some embodiments, the bladder cancer is transitional cell carcinomaor urothelial carcinoma (such as metastatic urothelial carcinoma),including, but not limited to, papillary tumors and flat carcinomas. Insome embodiments, the bladder cancer is metastatic urothelial carcinoma.In some embodiments, the bladder cancer is urothelial carcinoma of thebladder. In some embodiments, the bladder cancer is urothelial carcinomaof the ureter. In some embodiments, the bladder cancer is urothelialcarcinoma of the urethra. In some embodiments, the bladder cancer isurothelial carcinoma of the renal pelvis.

In some embodiments, the bladder cancer is squamous cell carcinoma. Insome embodiments, the bladder cancer is non-squamous cell carcinoma. Insome embodiments, the bladder cancer is adenocarcinoma. In someembodiments, the bladder cancer is small cell carcinoma.

In some embodiments, the bladder cancer is early stage bladder cancer,non-metastatic bladder cancer, non-invasive bladder cancer,non-muscle-invasive bladder cancer, primary bladder cancer, advancedbladder cancer, locally advanced bladder cancer (such as unresectablelocally advanced bladder cancer), metastatic bladder cancer, or bladdercancer in remission. In some embodiments, the bladder cancer islocalized resectable, localized unresectable, or unresectable. In someembodiments, the bladder cancer is a high grade, non-muscle-invasivecancer that has been refractory to standard intra-bladder infusion(intravesicular) therapy.

The methods provided herein can be used to treat an individual (e.g.,human) who has been diagnosed with or is suspected of having bladdercancer. In some embodiments, the individual is human. In someembodiments, the individual is at least about any of 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, or 85 years old. In some embodiments,the individual is male. In some embodiments, the individual is female.In some embodiments, the individual has undergone a tumor resection. Insome embodiments, the individual has refused surgery. In someembodiments, the individual is medically inoperable. In someembodiments, the individual is at a clinical stage of Ta, Tis, T1, T2,T3a, T3b, or T4 bladder cancer. In some embodiments, the individual isat a clinical stage of Tis, CIS, Ta, or T1.

In some embodiments, the individual is a human who exhibits one or moresymptoms associated with bladder cancer. In some embodiments, theindividual is at an early stage of bladder cancer. In some embodiments,the individual is at an advanced stage of bladder cancer. In some ofembodiments, the individual is genetically or otherwise predisposed(e.g., having a risk factor) to developing bladder cancer. Individualsat risk for bladder cancer include, e.g., those having relatives whohave experienced bladder cancer, and those whose risk is determined byanalysis of genetic or biochemical markers. In some embodiments, theindividual is positive for SPARC expression (for example based onimmunohistochemistry (IHC) standard). In some embodiments, theindividual is negative for SPARC expression. In some embodiments, theindividual has a mutation in FGFR2. In some embodiments, the individualhas a mutation in p53. In some embodiments, the individual has amutation in MIB-1. In some embodiments, the individual has a mutation inone or more of FEZ1/LZTS1, PTEN, CDKN2A/MTS1/P6, CDKN2B/INK4B/P15, TSC1,DBCCR1, HRAS1, ERBB2, or NF1. In some embodiments, the individual hasmutation in both p53 and PTEN.

The present invention for example provides in some embodiments methodsof treatment bladder cancer in an individual comprising administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a lumus drug),wherein the treatment is based on the mutation status of one or more ofFEZ1/LZTS1, PTEN, CDKN2A/MTS1/P6, CDKN2B/INK4B/P15, TSC1, DBCCR1, HRAS1,ERBB2, or NF1. In some embodiments, there is provided a method oftreating bladder cancer in an individual comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug), wherein theindividual is selected for treatment based on the mutation status of oneor more of FEZ1/LZTS1, PTEN, CDKN2A/MTS1/P6, CDKN2B/INK4B/P15, TSC1,DBCCR1, HRAS1, ERBB2, or NF1. In some embodiments, there is provided amethod of selecting (including identifying) an individual having bladdercancer for treating with a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug), wherein the methodcomprises determining the mutation status of one or more of FEZ1/LZTS1,PTEN, CDKN2A/MTS1/P6, CDKN2B/INK4B/P15, TSC1, DBCCR1, HRAS1, ERBB2, orNF1. The mutation status of one or more of FEZ1/LZTS1, PTEN,CDKN2A/MTS1/P6, CDKN2B/INK4B/P15, TSC1, DBCCR1, HRAS1, ERBB2, or NF1 canalso be useful for determining any of the following: (a) probable orlikely suitability of an individual to initially receive treatment(s);(b) probable or likely unsuitability of an individual to initiallyreceive treatment(s); (c) responsiveness to treatment; (d) probable orlikely suitability of an individual to continue to receive treatment(s);(e) probable or likely unsuitability of an individual to continue toreceive treatment(s); (f) adjusting dosage; (g) predicting likelihood ofclinical benefits.

In some embodiments, the individual has a partial or complete monosomy(such as monosomy 9). In some embodiments, the individual has a deletionin chromosome 11p. In some embodiments, the individual has a deletion inchromosome 13q. In some embodiments, the individual has a deletion inchromosome 17p. In some embodiments, the individual has a deletion inchromosome 1p. In some embodiments, the individual as a chromosome lossof 8p12-22.

In some embodiments, the individual overexpresses p73, c-myc, or cyclinD1.

The methods provided herein may be practiced in an adjuvant setting. Insome embodiments, the method is practiced in a neoadjuvant setting,i.e., the method may be carried out before the primary/definitivetherapy. In some embodiments, the method is used to treat an individualwho has previously been treated. In some embodiments, the individual hasnot previously been treated. In some embodiments, the method is used asa first line therapy. In some embodiments, the method is used as asecond line therapy.

In some embodiments, the individual has been previously treated forbladder cancer (also referred to as the “prior therapy”). In someembodiments, individual has been previously treated with a standardtherapy for bladder cancer. In some embodiments, the prior standardtherapy is treatment with BCG. In some embodiments, the prior standardtherapy is treatment with mitomycin C. In some embodiments, the priorstandard therapy is treatment with interferon (such as interferon-α). Insome embodiments, the individual has bladder cancer in remission,progressive bladder cancer, or recurrent bladder cancer. In someembodiments, the individual is resistant to treatment of bladder cancerwith other agents (such as platinum-based agents, BCG, mitomycin C, orinterferon). In some embodiments, the individual is initially responsiveto treatment of bladder cancer with other agents (such as platinum-basedagents, or BCG) but has progressed after treatment.

In some embodiments, the individual has recurrent bladder cancer (suchas a bladder cancer at the clinical stage of Ta, Tis, T1, T2, T3a, T3b,or T4) after a prior therapy (such as prior standard therapy, forexample treatment with BCG). For example, the individual may beinitially responsive to the treatment with the prior therapy, butdevelops bladder cancer after about any of about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 24, 36, 48, or 60 months upon the cessation of the priortherapy.

In some embodiments, the individual is refractory to a prior therapy(such as prior standard therapy, for example treatment with BCG).

In some embodiments, the individual has progressed on the prior therapy(such as prior standard therapy, for example treatment with BCG) at thetime of treatment. For example, the individual has progressed within anyof about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months upon treatmentwith the prior therapy.

In some embodiments, the individual is resistant to the prior therapy(such as prior standard therapy, for example treatment with BCG).

In some embodiments, the individual is unsuitable to continue with theprior therapy (such as prior standard therapy, for example treatmentwith BCG), for example due to failure to respond and/or due to toxicity.

In some embodiments, the individual is non-responsive to the priortherapy (such as prior standard therapy, for example treatment withBCG).

In some embodiments, the individual is partially responsive to the priortherapy (such as prior standard therapy, for example treatment withBCG), or exhibits a less desirable degree of responsiveness.

In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising intravesicularlyadministering (for example via urethral catheterization) to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising intravesicularly administering (forexample via urethral catheterization) to the individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor (such as a limus drug) and an albumin. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug) and an albumin, wherein the mTOR inhibitor (such as alimus drug) in the nanoparticles is coated with the albumin. In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising intravesicularly administering (forexample via urethral catheterization) to the individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor (such as a limus drug) and an albumin, wherein thenanoparticles have an average particle size of no greater than about 200nm (such as no greater than about 150 nm). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug) and an albumin, wherein the nanoparticles comprise themTOR inhibitor (such as a limus drug) coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug) and human albumin, wherein the nanoparticles comprisethe mTOR inhibitor (such as a limus drug) coated with the human albumin,wherein the nanoparticles have an average particle size of no greaterthan about 150 nm (such as no greater than about 120 nm, for exampleabout 100 nm), wherein the weight ratio of human albumin and the mTORinhibitor (such as a limus drug) in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, the mTORinhibitor (i.e., the mTOR inhibitor in the mTOR nanoparticlecomposition) is administered at a dose of about 5 mg to about 500 mg(including for example about 30 mg to about 400 mg, such as about 100mg). In some embodiments, the limus nanoparticle composition isadministered weekly. In some embodiments, the composition isadministered weekly for 6 weeks, optionally followed by monthlymaintenance thereafter.

In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising intravesicularlyadministering (for example via urethral catheterization) to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising intravesicularly administering (forexample via urethral catheterization) to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin. In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising intravesicularly administering (forexample via urethral catheterization) to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles have an average particle sizeof no greater than about 200 nm (such as no greater than about 150 nm).In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising intravesicularlyadministering (for example via urethral catheterization) to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, and wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising intravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising sirolimus and human albumin, whereinthe nanoparticles comprise sirolimus coated with the human albumin,wherein the nanoparticles have an average particle size of no greaterthan about 150 nm (such as no greater than about 120 nm, for exampleabout 100 nm), wherein the weight ratio of human albumin and sirolimusin the composition is about 9:1 or less (such as about 9:1 or about8:1). In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising Nab-sirolimus. In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising intravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of Nab-sirolimus.In some embodiments, the limus drug (i.e., limus drug in the limusnanoparticle composition) is administered at a dose of about 5 mg toabout 500 mg (including for example about 30 mg to about 400 mg, such asabout 100 mg). In some embodiments, the limus nanoparticle compositionis administered weekly. In some embodiments, the composition isadministered weekly for 6 weeks, optionally followed by monthlymaintenance thereafter. In some embodiments, the composition isadministered with about 30 minutes to about 4 hours, such as about 1hour to about 2 hours of retention in the bladder.

In some embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug). In some embodiments, there is provided a method of treatingnon-muscle invasive bladder cancer in an individual (e.g., human)comprising administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, there is provided a method of treatingnon-muscle invasive bladder cancer in an individual (e.g., human)comprising administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin. In some embodiments, there is provided a method of treatingnon-muscle invasive bladder cancer in an individual (e.g., human)comprising administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments,there is provided a method of treating non-muscle invasive bladdercancer in an individual (e.g., human) comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising sirolimus and human albumin, wherein the nanoparticlescomprise sirolimus coated with the human albumin, wherein thenanoparticles have an average particle size of no greater than about 150nm (such as no greater than about 120 nm, for example about 100 nm),wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingadministering to the individual an effective amount of a compositioncomprising Nab-sirolimus. In some embodiments, there is provided amethod of treating non-muscle invasive bladder cancer in an individual(e.g., human) comprising administering to the individual an effectiveamount of Nab-sirolimus. In some embodiments, the limus drug isadministered at a dose of about 5 mg to about 500 mg (including forexample about 30 mg to about 400 mg, such as about 100 mg). In someembodiments, the limus nanoparticle composition is administered weekly.In some embodiments, the composition is administered weekly for 6 weeks,optionally followed by monthly maintenance thereafter.

In some embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug). In some embodiments, there is provided a method of treatingnon-muscle invasive bladder cancer in an individual (e.g., human)comprising intravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, there is provided a method of treating non-muscle invasivebladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, there is provided a method of treating non-muscle invasivebladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments,there is provided a method of treating non-muscle invasive bladdercancer in an individual (e.g., human) comprising intravesicularlyadministering (for example via urethral catheterization) to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, and wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm). In some embodiments, there is provided amethod of treating non-muscle invasive bladder cancer in an individual(e.g., human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, there is provided a method of treatingnon-muscle invasive bladder cancer in an individual (e.g., human)comprising intravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising Nab-sirolimus. In some embodiments, there is provided amethod of treating non-muscle invasive bladder cancer in an individual(e.g., human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount ofNab-sirolimus. In some embodiments, the limus drug is administered at adose of about 5 mg to about 500 mg (including for example about 30 mg toabout 400 mg, such as about 100 mg). In some embodiments, the limusnanoparticle composition is administered weekly. In some embodiments,the composition is administered weekly for 6 weeks, optionally followedby monthly maintenance thereafter.

In some embodiments, there is provided a method of treatingBCG-refractory bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug). In some embodiments, there is provided a method of treatingBCG-refractory bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, there is provided a method of treating BCG-refractorybladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, there is provided a method of treating BCG-refractorybladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments,there is provided a method of treating BCG-refractory bladder cancer inan individual (e.g., human) comprising intravesicularly administering(for example via urethral catheterization) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles comprise a limusdrug coated with albumin, and wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, there is provided a method oftreating BCG-refractory bladder cancer in an individual (e.g., human)comprising intravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising nanoparticles comprising sirolimus and human albumin, whereinthe nanoparticles comprise sirolimus coated with the human albumin,wherein the nanoparticles have an average particle size of no greaterthan about 150 nm (such as no greater than about 120 nm, for exampleabout 100 nm), wherein the weight ratio of human albumin and sirolimusin the composition is about 9:1 or less (such as about 9:1 or about8:1). In some embodiments, there is provided a method of treatingBCG-refractory bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual an effective amount of a compositioncomprising Nab-sirolimus. In some embodiments, there is provided amethod of treating BCG-refractory bladder cancer in an individual (e.g.,human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual an effective amount ofNab-sirolimus. In some embodiments, the limus drug is administered at adose of about 5 mg to about 500 mg (including for example about 30 mg toabout 400 mg, such as about 100 mg). In some embodiments, the limusnanoparticle composition is administered weekly. In some embodiments,the composition is administered weekly for 6 weeks, optionally followedby monthly maintenance thereafter.

In some embodiments, there is provided a method of treating metastaticbladder cancer (such as metastatic urothelial carcinoma) in anindividual, comprising intravenously administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (such as a limus drug). In some embodiments, there isprovided a method of treating metastatic bladder cancer (such asmetastatic urothelial carcinoma) in an individual, comprisingintravenously administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, there is provided a method of treatingmetastatic bladder cancer (such as metastatic urothelial carcinoma) inan individual, comprising intravenously administering to the individualan effective amount of a composition comprising nanoparticles comprisinga limus drug coated with albumin. In some embodiments, there is provideda method of treating metastatic bladder cancer (such as metastaticurothelial carcinoma) in an individual, comprising intravenouslyadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and albumin and havingan average diameter of no greater than about 200 nm. In someembodiments, there is provided a method of treating metastatic bladdercancer (such as metastatic urothelial carcinoma) in an individual,comprising intravenously administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugcoated with albumin and having an average diameter of no greater thanabout 200 nm. In some embodiments, there is provided a method oftreating metastatic bladder cancer (such as metastatic urothelialcarcinoma) in an individual, comprising intravenously administering tothe individual an effective amount of a composition comprisingnanoparticles comprising sirolimus coated with human albumin and havingan average diameter of no greater than about 150 (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, there is provideda method of treating metastatic bladder cancer (such as metastaticurothelial carcinoma) in an individual, comprising intravenouslyadministering to the individual an effective amount of a compositioncomprising Nab-sirolimus. In some embodiments, there is provided amethod of treating metastatic bladder cancer (such as metastaticurothelial carcinoma) in an individual, comprising intravenouslyadministering to the individual an effective amount of Nab-sirolimus. Insome embodiments, the treatment is second line treatment.

In some embodiments, there is provided a method of treating aplatinum-refractory bladder cancer (such as metastaticplatinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (such as a limus drug). In some embodiments, there isprovided a method of treating a platinum-refractory bladder cancer (suchas metastatic platinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin. In some embodiments, there is provided amethod of treating platinum-refractory bladder cancer (such asmetastatic platinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug coated with albumin. In some embodiments, there is provided amethod of treating platinum-refractory bladder cancer (such asmetastatic platinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and albumin and having an average diameter of no greater thanabout 200 nm. In some embodiments, there is provided a method oftreating platinum-refractory bladder cancer (such as metastaticplatinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug coated with albumin and having an average diameter of nogreater than about 200 nm. In some embodiments, there is provided amethod of treating platinum-refractory bladder cancer (such asmetastatic platinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising nanoparticles comprisingsirolimus coated with human albumin and having an average diameter of nogreater than about 200 nm, wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, there is provided a method of treatingplatinum-refractory bladder cancer (such as metastaticplatinum-refractory bladder cancer, for example metastaticplatinum-refractory urothelial carcinoma) in an individual, comprisingadministering (such as intravenously administering) to the individual aneffective amount of a composition comprising Nab-sirolimus. In someembodiments, there is provided a method of treating platinum-refractorybladder cancer (such as metastatic platinum-refractory bladder cancer,for example metastatic platinum-refractory urothelial carcinoma) in anindividual, comprising administering (such as intravenouslyadministering) to the individual an effective amount of Nab-sirolimus.

The methods described herein are useful for various aspects of bladdercancer treatment. In some embodiments, there is provided a method ofinhibiting bladder cancer cell proliferation (such as bladder cancertumor growth) in an individual, comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin. In some embodiments, at leastabout 10% (including for example at least about any of 20%, 30%, 40%,60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. In someembodiments, the limus drug is sirolimus. In some embodiments, the limusdrug in the nanoparticle in the composition is administered byintravenous administration. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravesicularadministration. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin. In someembodiments, the composition comprises nanoparticles having an averagediameter of no greater than about 200 nm. In some embodiments, thecomposition comprises nanoparticles comprising a limus drug coated withalbumin, wherein the nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the compositioncomprises nanoparticles comprising sirolimus coated with human albumin,wherein the nanoparticles have an average diameter of no greater thanabout 150 nm (such as no greater than about 120 nm, for example about100 nm), wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, the composition comprises Nab-sirolimus. In someembodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of preventing localrecurrence (e.g., recurrence of tumor after resection) in an individualhaving bladder cancer, comprising administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin. In some embodiments, at least about 10%(including for example at least about any of 20%, 30%, 40%, 60%, 70%,80%, 90%, or 100%) metastasis is inhibited. In some embodiments, thelimus drug is sirolimus. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravenousadministration. In some embodiments, the limus drug in the nanoparticlein the composition is administered by intravesicular administration. Insome embodiments, the composition comprises nanoparticles comprising alimus drug coated with albumin. In some embodiments, the compositioncomprises nanoparticles having an average diameter of no greater thanabout 200 nm. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin, wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the composition comprises nanoparticles comprisingsirolimus coated with human albumin, wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, the compositioncomprises Nab-sirolimus. In some embodiments, the composition isNab-sirolimus.

In some embodiments, there is provided a method of inhibiting bladdercancer tumor metastasis in an individual, comprising administering tothe individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin. In someembodiments, at least about 10% (including for example at least aboutany of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis isinhibited. In some embodiments, a method of inhibiting metastasis tolymph node is provided. In some embodiments, a method of inhibitingmetastasis to the lung is provided. In some embodiments, the limus drugis sirolimus. In some embodiments, the limus drug in the nanoparticle inthe composition is administered by intravenous administration. In someembodiments, the limus drug in the nanoparticle in the composition isadministered by intravesicular administration. In some embodiments, thecomposition comprises nanoparticles comprising a limus drug coated withalbumin. In some embodiments, the composition comprises nanoparticleshaving an average diameter of no greater than about 200 nm. In someembodiments, the composition comprises nanoparticles comprising a limusdrug coated with albumin, wherein the nanoparticles have an averagediameter of no greater than about 200 nm. In some embodiments, thecomposition comprises nanoparticles comprising sirolimus coated withhuman albumin, wherein the nanoparticles have an average diameter of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of reducing (such aseradiating) pre-existing bladder cancer tumor metastasis (such aspulmonary metastasis or metastasis to the lymph node) in an individual,comprising administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, at least about 10% (including for exampleat least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%)metastasis is reduced. In some embodiments, a method of reducingmetastasis to lymph node is provided. In some embodiments, a method ofreducing metastasis to the lung is provided. In some embodiments, thelimus drug is sirolimus. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravenousadministration. In some embodiments, the limus drug in the nanoparticlein the composition is administered by intravesicular administration. Insome embodiments, the composition comprises nanoparticles comprising alimus drug coated with albumin. In some embodiments, the compositioncomprises nanoparticles having an average diameter of no greater thanabout 200 nm. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin, wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the composition comprises nanoparticles comprisingsirolimus coated with human albumin, wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, the compositioncomprises Nab-sirolimus. In some embodiments, the composition isNab-sirolimus.

In some embodiments, there is provided a method of reducing incidence orburden of preexisting bladder cancer tumor metastasis (such as pulmonarymetastasis or metastasis to the lymph node) in an individual, comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the limus drug is sirolimus. In some embodiments, the limusdrug in the nanoparticle in the composition is administered byintravenous administration. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravesicularadministration. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin. In someembodiments, the composition comprises nanoparticles having an averagediameter of no greater than about 200 nm. In some embodiments, thecomposition comprises nanoparticles comprising a limus drug coated withalbumin, wherein the nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the compositioncomprises nanoparticles comprising sirolimus coated with human albumin,wherein the nanoparticles have an average diameter of no greater thanabout 150 nm (such as no greater than about 120 nm, for example about100 nm), wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, the composition comprises Nab-sirolimus. In someembodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of reducing bladdercancer tumor size in an individual, comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin. In some embodiments, the tumorsize is reduced at least about 10% (including for example at least aboutany of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments,the limus drug is sirolimus. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravenousadministration. In some embodiments, the limus drug in the nanoparticlein the composition is administered by intravesicular administration. Insome embodiments, the composition comprises nanoparticles comprising alimus drug coated with albumin. In some embodiments, the compositioncomprises nanoparticles having an average diameter of no greater thanabout 200 nm. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin, wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the composition comprises nanoparticles comprisingsirolimus coated with human albumin, wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1).

In some embodiments, there is provided a method of prolonging time todisease progression of bladder cancer in an individual, comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the method prolongs the time to disease progression by atleast any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In someembodiments, the limus drug is sirolimus. In some embodiments, the limusdrug in the nanoparticle in the composition is administered byintravenous administration. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravesicularadministration. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin. In someembodiments, the composition comprises nanoparticles having an averagediameter of no greater than about 200 nm. In some embodiments, thecomposition comprises nanoparticles comprising a limus drug coated withalbumin, wherein the nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the compositioncomprises nanoparticles comprising sirolimus coated with human albumin,wherein the nanoparticles have an average diameter of no greater thanabout 150 nm (such as no greater than about 120 nm, for example about100 nm), wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, the composition comprises Nab-sirolimus. In someembodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of prolonging survivalof an individual having bladder cancer, comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin. In some embodiments, the methodprolongs the survival of the individual by at least any of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 month. In some embodiments, thelimus drug is sirolimus. In some embodiments, the limus drug in thenanoparticle in the composition is administered by intravenousadministration. In some embodiments, the limus drug in the nanoparticlein the composition is administered by intravesicular administration. Insome embodiments, the composition comprises nanoparticles comprising alimus drug coated with albumin. In some embodiments, the compositioncomprises nanoparticles having an average diameter of no greater thanabout 200 nm. In some embodiments, the composition comprisesnanoparticles comprising a limus drug coated with albumin, wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the composition comprises nanoparticles comprisingsirolimus coated with human albumin, wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, the compositioncomprises Nab-sirolimus. In some embodiments, the composition isNab-sirolimus.

In some embodiments, there is provided a method of alleviating one ormore symptoms in an individual having bladder cancer, comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the limus drug in the nanoparticle in the composition isadministered by intravenous administration. In some embodiments, thelimus drug in the nanoparticle in the composition is administered byintravesicular administration. In some embodiments, the compositioncomprises nanoparticles comprising a limus drug coated with albumin. Insome embodiments, the composition comprises nanoparticles having anaverage diameter of no greater than about 200 nm. In some embodiments,the composition comprises nanoparticles comprising a limus drug coatedwith albumin, wherein the nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the compositioncomprises nanoparticles comprising sirolimus coated with human albumin,wherein the nanoparticles have an average diameter of no greater thanabout 150 nm (such as no greater than about 120 nm, for example about100 nm), wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, the composition comprises Nab-sirolimus. In someembodiments, the composition is Nab-sirolimus.

In some embodiments, there is provided a method of suppression theprogression of CIS (carcinoma in situ) lesions in an individual havingbladder cancer, comprising administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin. In some embodiments, the limus drug in the nanoparticlein the composition is administered by intravenous administration. Insome embodiments, the limus drug in the nanoparticle in the compositionis administered by intravesicular administration. In some embodiments,the composition comprises nanoparticles comprising a limus drug coatedwith albumin. In some embodiments, the composition comprisesnanoparticles having an average diameter of no greater than about 200nm. In some embodiments, the composition comprises nanoparticlescomprising a limus drug coated with albumin, wherein the nanoparticleshave an average diameter of no greater than about 200 nm. In someembodiments, the composition comprises nanoparticles comprisingsirolimus coated with human albumin, wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (such as no greaterthan about 120 nm, for example about 100 nm), wherein the weight ratioof human albumin and sirolimus in the composition is about 9:1 or less(such as about 9:1 or about 8:1). In some embodiments, the compositioncomprises Nab-sirolimus. In some embodiments, the composition isNab-sirolimus.

Also provided are pharmaceutical compositions comprising nanoparticlescomprising an mTOR inhibitor (such as limus drug, for example sirolimus)for use in any of the methods of treating bladder cancer describedherein. In some embodiments, the compositions comprise nanoparticlescomprising an mTOR inhibitor (such as limus drug, for example sirolimus)and albumin (such as human albumin).

Methods of Combination Therapy

The present invention also provides combination therapy methods fortreating bladder cancer. Thus, in some embodiments, the individual beingtreated with the mTOR nanoparticle composition is also subjected to asecond therapy. In some embodiments, the second therapy is surgery,radiation, immunotherapy, and/or chemotherapy. It is understood thatreference to and description of methods of treating bladder cancer aboveis exemplary and that the description applies equally to and includesmethods of treating bladder cancer using combination therapy.

In some embodiments, the method comprises administering the mTORinhibitor nanoparticle composition (such as a limus nanoparticlecomposition) and at least another agent. In some embodiments, thenanoparticle composition and the other agent (including the specifictherapeutic agents described herein) are administered simultaneously.When the drugs are administered simultaneously, the drug in thenanoparticles and the other agent may be contained in the samecomposition (e.g., a composition comprising both the nanoparticles andthe other agent) or in separate compositions (e.g., the nanoparticlesare contained in one composition and the other agent is contained inanother composition). In some embodiments, the nanoparticle compositionand the other agent are administered sequentially. Either thenanoparticle composition or the other agent may be administered first.The nanoparticle composition and the other agent are contained inseparate compositions, which may be contained in the same or differentpackages. In some embodiments, the administration of the nanoparticlecomposition and the other agent are concurrent, i.e., the administrationperiod of the nanoparticle composition and that of the other agentoverlap with each other.

In some embodiments, the method comprises administration of an mTORnanoparticle composition (such as a limus nanoparticle composition) incombination with an immunotherapy (such as administration of animmunotherapeutic agent). Suitable immunotherapeutic agents that can becombined with the mTOR nanoparticle composition (such as a limusnanoparticle composition) include, but are not limited to, BCG,interferons, and other immune stimulatory cytokines.

Thus, the present application in some embodiments provides a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug); and (b) an immunotherapeutic agent. Thus, thepresent application in some embodiments provides a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin; and (b) an immunotherapeutic agent. In some embodiments, thereis provided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug in the nanoparticlesis coated with the albumin; and (b) an immunotherapeutic agent. In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm); and(b) an immunotherapeutic agent. In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles comprise a limus drug coatedwith albumin, and wherein the nanoparticles have an average particlesize of no greater than about 200 nm (such as no greater than about 150nm); and (b) an immunotherapeutic agent. In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with the human albumin, wherein the nanoparticles havean average particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1); and (b) an immunotherapeuticagent. In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising Nab-sirolimus; and (b) an immunotherapeuticagent. In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) administering to the individual (a) an effectiveamount of Nab-sirolimus; and (b) an immunotherapeutic agent. In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

In some embodiments, the other agent is BCG (Bacillus Calmette-Guérin),a live attenuated form of Mycobacterium bovis. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a limus drug); and(b) an effective amount of BCG. In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin; and (b) an effective amount of BCG. In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; and (b) an effective amount of BCG. In some embodiments, thereis provided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of BCG. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, and wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm); and (b) an effective amount of BCG. Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1); and (b) an effective amount of BCG. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingNab-sirolimus; and (b) an effective amount of BCG. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) administeringto the individual (a) an effective amount of Nab-sirolimus; and (b) aneffective amount of BCG. In some embodiments, the bladder cancer isnon-muscle invasive bladder cancer. In some embodiments, the bladdercancer is BCG-refractory bladder cancer. In some embodiments, thebladder cancer is BCG-refractory non-muscle invasive bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer (such as non-muscle invasive bladder cancer) in an individual(e.g., human) comprising intravesicularly administering (for example viaurethral catheterization) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the dose of the limus drug in the composition is about5 mg to about 500 mg (such as about 30 mg to about 400 mg, for exampleabout 100 mg); and (b) an effective amount of BCG, wherein the dose ofBCG is about 8 mg to about 100 mg (such as about 25 mg to about 85 mg,for example about 80 mg). In some embodiments, there is provided amethod of treating bladder cancer (such as non-muscle invasive bladdercancer) in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin, wherein the dose of the limus drug in the composition is about5 mg to about 500 mg (such as about 30 to about 400 mg, for exampleabout 100 mg); and (b) an effective amount of BCG, wherein the dose ofBCG is about 8 mg to about 100 mg (such as about 25 mg to about 85 mg,for example about 80 mg). In some embodiments, there is provided amethod of treating bladder cancer (such as non-muscle invasive bladdercancer) in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm),wherein the dose of the limus drug in the composition is about 5 mg toabout 500 mg (such as about 30 mg to about 400 mg, for example about 100mg); and (b) an effective amount of BCG, wherein the dose of BCG isabout 8 mg to about 100 mg (such as about 25 mg to about 85 mg, forexample about 80 mg). In some embodiments, there is provided a method oftreating bladder cancer (such as non-muscle invasive bladder cancer) inan individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm), wherein the dose of thelimus drug in the composition is about 5 mg to about 500 mg (such asabout 30 mg to about 400 mg, for example about 100 mg); and (b) aneffective amount of BCG, wherein the dose of BCG is about 8 mg to about100 mg (such as about 25 mg to about 85 mg, for example about 80 mg). Insome embodiments, there is provided a method of treating bladder cancer(such as non-muscle invasive bladder cancer) in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with the human albumin, wherein the nanoparticles havean average particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1) wherein the dose of the sirolimusin the composition is about 5 mg to about 500 mg (such as about 30 mg toabout 400 mg, for example about 100 mg); and (b) an effective amount ofBCG, wherein the dose of BCG is about 8 mg to about 100 mg (such asabout 25 mg to about 85 mg, for example about 80 mg). In someembodiments, there is provided a method of treating bladder cancer (suchas non-muscle invasive bladder cancer) in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising Nab-sirolimus, wherein thedose of the limus drug in the composition is about 5 mg to about 500 mg(such as about 30 to about 400 mg, for example about 100 mg); and (b) aneffective amount of BCG, wherein the dose of BCG is about 8 mg to about100 mg (such as about 25 mg to about 85 mg, for example about 80 mg). Insome embodiments, there is provided a method of treating bladder cancer(such as non-muscle invasive bladder cancer) in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus, wherein the dose ofthe limus drug in the composition is about 5 mg to about 500 mg (such asabout 30 to about 400 mg, for example about 100 mg); and (b) aneffective amount of BCG, wherein the dose of BCG is about 8 mg to about100 mg (such as about 25 mg to about 85 mg, for example about 80 mg). Insome embodiments, the bladder cancer is non-muscle invasive bladdercancer. In some embodiments, the bladder cancer is BCG-refractorybladder cancer. In some embodiments, the bladder cancer isBCG-refractory non-muscle invasive bladder cancer.

In some embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1), wherein the dose of the sirolimus in the composition isabout 5 mg to about 500 mg (such as about 30 mg to about 400 mg, forexample about 100 mg) weekly; and (b) an effective amount of BCG,wherein the dose of BCG is about 8 mg to about 100 mg (such as about 25mg to about 85 mg, for example about 80 mg) weekly. In some embodiments,there is provided a method of treating bladder cancer (such asnon-muscle invasive bladder cancer) in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising Nab-sirolimus, wherein thedose of the limus drug in the composition is about 5 mg to about 500 mg(such as about 30 mg to about 400 mg, for example about 100 mg) weekly;and (b) an effective amount of BCG, wherein the dose of BCG is about 8mg to about 100 mg (such as about 25 mg to about 85 mg, for exampleabout 80 mg) weekly. In some embodiments, there is provided a method oftreating bladder cancer (such as non-muscle invasive bladder cancer) inan individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) administering to the individual (a) an effectiveamount of Nab-sirolimus, wherein the dose of the limus drug in thecomposition is about 5 mg to about 500 mg (such as about 30 mg to about400 mg, for example about 100 mg) weekly; and (b) an effective amount ofBCG, wherein the dose of BCG is about 8 mg to about 100 mg (such asabout 25 mg to about 85 mg, for example about 80 mg) weekly. In someembodiments, the nanoparticle composition and/or BCG are provided in avolume of about 20-ml to about 150 ml (such as about 50 ml). In someembodiments, the nanoparticle composition and/or BCG are retained in thebladder for about 30 minutes to about 4 hours (such as about 30minutes).

In some embodiments, the methods of combination therapy described hereincomprise administration of an interferon, such as interferon a, with orwithout BCG.

Thus, for example, in some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug); (b) an effective amount of an interferon (suchas interferon α). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin; (b) an effective amount of an interferon (such as interferonα). In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; (b) an effective amount of an interferon (such as interferonα). In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm); (b) aneffective amount of an interferon (such as interferon α). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm); (b) an effective amountof an interferon (such as interferon a). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with the human albumin, wherein the nanoparticles havean average particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1); (b) an effective amount of aninterferon (such as interferon α). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising Nab-sirolimus; (b) aneffective amount of an interferon (such as interferon α). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) administering to the individual (a) an effectiveamount of Nab-sirolimus; (b) an effective amount of an interferon (suchas interferon α). In some embodiments, the bladder cancer is non-muscleinvasive bladder cancer. In some embodiments, the bladder cancer isBCG-refractory bladder cancer. In some embodiments, the bladder canceris BCG-refractory non-muscle invasive bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug); (b) an effective amount of BCG; and (c) an effectiveamount of an interferon (such as interferon α). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin; (b) an effectiveamount of BCG; and (c) an effective amount of an interferon (such asinterferon α). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; (b) an effective amount of BCG; and (c) an effective amount ofan interferon (such as interferon α). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); (b) an effective amount of BCG; and (c) an effectiveamount of an interferon (such as interferon α). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, and wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm); (b) an effective amount of BCG; and (c)an effective amount of an interferon (such as interferon α). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1); (b) an effective amount of BCG; and (c) an effective amountof an interferon (such as interferon α). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising Nab-sirolimus; (b) aneffective amount of BCG; and (c) an effective amount of an interferon(such as interferon α). In some embodiments, there is provided a methodof treating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) administering to the individual (a) aneffective amount of Nab-sirolimus; (b) an effective amount of BCG; and(c) an effective amount of an interferon (such as interferon α). In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer in an individual, comprising administering to the individual a)an effective amount of at least another therapeutic agent. In someembodiments, the other therapeutic agent is selected from the groupconsisting of an alkylating agent, an anthracycline antibiotic, a DNAcrosslinking agent, an antimetabolite, an indolequinone, a taxane, or aplatinum-based agent. In some embodiments, the other therapeutic agentis selected from the group consisting of mitomycin, epirubicin,doxorubicin, valrubicin, gemcitabine, apaziquone, docetaxel, paclitaxel,and cisplatin.

In some embodiments, the other agent to be administered in combinationwith the mTOR nanoparticle composition (such as the limus nanoparticlecomposition) is an alkylating agent. Thus, for example, in someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug); and (b) an effective amount of an alkylating agent(such as mitomycin). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin; and (b) an effective amount of an alkylating agent (such asmitomycin). In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; and (b) an effective amount of an alkylating agent (such asmitomycin). In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm); and(b) an effective amount of an alkylating agent (such as mitomycin). Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm); and (b) an effectiveamount of an alkylating agent (such as mitomycin). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising sirolimus and human albumin, wherein thenanoparticles comprise sirolimus coated with the human albumin, whereinthe nanoparticles have an average particle size of no greater than about150 nm (such as no greater than about 120 nm, for example about 100 nm),wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1); and(b) an effective amount of an alkylating agent (such as mitomycin). Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising Nab-sirolimus; and (b) an effective amount of analkylating agent (such as mitomycin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus; and (b) aneffective amount of an alkylating agent (such as mitomycin). In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

In some embodiments, the other agent to be administered in combinationwith the mTOR nanoparticle composition (such as the limus nanoparticlecomposition) is a DNA crosslinking agent. Thus, for example, in someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug); and (b) an effective amount of a DNA crosslinkingagent (such as mitomycin). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin; and (b) an effective amount of a DNAcrosslinking agent (such as mitomycin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug in the nanoparticlesis coated with the albumin; and (b) an effective amount of a DNAcrosslinking agent (such as mitomycin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of a DNA crosslinking agent(such as mitomycin). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles comprise a limus drug coated withalbumin, and wherein the nanoparticles have an average particle size ofno greater than about 200 nm (such as no greater than about 150 nm); and(b) an effective amount of a DNA crosslinking agent (such as mitomycin).In some embodiments, there is provided a method of treating bladdercancer in an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1); and (b) an effective amount of a DNA crosslinking agent(such as mitomycin). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising Nab-sirolimus; and (b) an effective amount of aDNA crosslinking agent (such as mitomycin). In some embodiments, thereis provided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus; and (b) aneffective amount of an alkylating agent (such as mitomycin). In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

In some embodiments, there is provided a method of treating non-muscleinvasive bladder cancer in an individual (e.g., human) comprisingintravesicularly administering (for example via urethralcatheterization) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1), wherein the dose of the sirolimus in the composition isabout 5 mg to about 500 mg (such as about 30 mg to about 400 mg, forexample about 100 mg) weekly; and (b) an effective amount of analkylating agent (such as mitomycin), wherein the dose of the alkylatingagent (such as mitomycin) is about 8 mg to about 100 mg (such as about25 mg to about 50 mg, for example about 40 mg) weekly. In someembodiments, there is provided a method of treating bladder cancer (suchas non-muscle invasive bladder cancer) in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising Nab-sirolimus, wherein thedose of the limus drug in the composition is about 5 mg to about 500 mg(such as about 30 mg to about 400 mg, for example about 100 mg) weekly;and (b) an effective amount of an alkylating agent (such as mitomycin),wherein the dose of the alkylating agent (such as mitomycin) is about 8mg to about 100 mg (such as about 25 mg to about 50 mg, for exampleabout 40 mg) weekly. In some embodiments, there is provided a method oftreating bladder cancer (such as non-muscle invasive bladder cancer) inan individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) administering to the individual (a) an effectiveamount of Nab-sirolimus, wherein the dose of the limus drug in thecomposition is about 5 mg to about 500 mg (such as about 30 mg to about400 mg, for example about 100 mg) weekly; and (b) an effective amount ofan alkylating agent (such as mitomycin), wherein the dose of thealkylating agent (such as mitomycin) is about 8 mg to about 100 mg (suchas about 25 mg to about 50 mg, for example about 40 mg) weekly. In someembodiments, the nanoparticle composition and/or the alkylating agent(such as mitomycin) are provided in a volume of about 20-ml to about 150ml (such as about 50 ml). In some embodiments, the nanoparticlecomposition and/or BCG are retained in the bladder for about 30 minutesto about 4 hours (such as about 30 minutes). In some embodiments, thebladder cancer is non-muscle invasive bladder cancer. In someembodiments, the bladder cancer is BCG-refractory bladder cancer. Insome embodiments, the bladder cancer is BCG-refractory non-muscleinvasive bladder cancer.

In some embodiments, the other agent is an anthracycline antibiotic.Thus, for example, in some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug); and (b) an effective amount of an anthracycline(such as epirubicin and/or doxorubicin, or valrubicin). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin; and (b) an effective amount of an anthracycline (such asepirubicin and/or doxorubicin, or valrubicin). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin, wherein the limusdrug in the nanoparticles is coated with the albumin; and (b) aneffective amount of an anthracycline (such as epirubicin and/ordoxorubicin, or valrubicin). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of an anthracycline (such asepirubicin and/or doxorubicin, or valrubicin). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, and wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm); and (b) an effective amount of ananthracycline (such as epirubicin and/or doxorubicin, or valrubicin). Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1); and (b) an effective amount of an anthracycline (such asepirubicin and/or doxorubicin, or valrubicin). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingNab-sirolimus; and (b) an effective amount of an anthracycline (such asepirubicin and/or doxirubicin). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) administering to the individual(a) an effective amount of Nab-sirolimus; and (b) an effective amount ofan anthracycline (such as epirubicin and/or doxorubicin, or valrubicin).In some embodiments, the bladder cancer is non-muscle invasive bladdercancer. In some embodiments, the bladder cancer is BCG-refractorybladder cancer. In some embodiments, the bladder cancer isBCG-refractory non-muscle invasive bladder cancer.

In some embodiments, the other agent is an antimetabolite. Thus, forexample, in some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin; and (b) an effective amount of an antimetabolite (such asgemcitabine). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug); and (b) an effective amount of an antimetabolite(such as gemcitabine). In some embodiments, there is provided a methodof treating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; and (b) an effective amount of an antimetabolite (such asgemcitabine). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm); and(b) an effective amount of an antimetabolite (such as gemcitabine). Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm); and (b) an effectiveamount of an antimetabolite (such as gemcitabine). In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising sirolimus and human albumin, wherein thenanoparticles comprise sirolimus coated with the human albumin, whereinthe nanoparticles have an average particle size of no greater than about150 nm (such as no greater than about 120 nm, for example about 100 nm),wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1); and(b) an effective amount of an antimetabolite (such as gemcitabine). Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising Nab-sirolimus; and (b) an effective amount of anantimetabolite (such as gemcitabine). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus; and (b) aneffective amount of an antimetabolite (such as gemcitabine). In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer. In some embodiments, theantimetabolite (such as gemcitabine) is administered weekly, for exampleby weekly intravesicular administration at the dose of 2 grams in 50 ml,each with about 30 minutes to about 4 hours, for example about 1 hour toabout 2 hours of retention time in the bladder. In some embodiments, theantimetabolite (such as gemcitabine) is administered immediately beforeor after the administration of the nanoparticle composition.

In some embodiments, the other agent is a taxane. Suitable taxanesinclude, but are not limited to, paclitaxel and docetaxel. The taxanecan be provided in nanoparticle forms. In some embodiments, the secondagent is Abraxane®. Thus, for example, in some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (such as a limus drug); and (b) an effective amount of ataxane (such as paclitaxel or docetaxel). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin; and (b) an effective amount of a taxane (suchas paclitaxel or docetaxel). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug in the nanoparticlesis coated with the albumin; and (b) an effective amount of a taxane(such as paclitaxel or docetaxel). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of a taxane (such aspaclitaxel or docetaxel). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles comprise a limus drug coatedwith albumin, and wherein the nanoparticles have an average particlesize of no greater than about 200 nm (such as no greater than about 150nm); and (b) an effective amount of a taxane (such as paclitaxel ordocetaxel). In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with thehuman albumin, wherein the nanoparticles have an average particle sizeof no greater than about 150 nm (such as no greater than about 120 nm,for example about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1); and (b) an effective amount of a taxane (such as paclitaxelor docetaxel). In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising Nab-sirolimus; and (b) an effective amount of ataxane (such as paclitaxel or docetaxel). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus; and (b) aneffective amount of a taxane (such as paclitaxel or docetaxel). In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

In some embodiments, the other agent is a platinum-based agent. Thus,for example, in some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug); and (b) an effective amount of a platinum-based agent(such as carboplatin or cisplatin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin; and (b) an effective amount of aplatinum-based agent (such as carboplatin or cisplatin). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; and (b) an effective amount of a platinum-based agent (such ascarboplatin or cisplatin). In some embodiments, there is provided amethod of treating bladder cancer in an individual (e.g., human)comprising administering (such as intravesicularly administering (forexample via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of a platinum-based agent(such as carboplatin or cisplatin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles comprise a limusdrug coated with albumin, and wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm); and (b) an effective amount of a platinum-based agent(such as carboplatin or cisplatin). In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) to the individual (a) aneffective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with the human albumin, wherein the nanoparticles havean average particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1); and (b) an effective amount of aplatinum-based agent (such as carboplatin or cisplatin). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising Nab-sirolimus; and (b) an effective amount of aplatinum-based agent (such as carboplatin or cisplatin). In someembodiments, there is provided a method of treating bladder cancer in anindividual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) administering to the individual (a) an effectiveamount of Nab-sirolimus; and (b) an effective amount of a platinum-basedagent (such as carboplatin or cisplatin). In some embodiments, thebladder cancer is non-muscle invasive bladder cancer. In someembodiments, the bladder cancer is BCG-refractory bladder cancer. Insome embodiments, the bladder cancer is BCG-refractory non-muscleinvasive bladder cancer.

The methods described herein may comprise administration of a limusnanoparticle composition in combination with two or more other agents.These two or more other agents may be of the same or different classes.For example, in some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (suchas a limus drug); (b) an effective amount of BCG; and (c) an effectiveamount of mitomycin. In some embodiments, there is provided a method oftreating bladder cancer in an individual (e.g., human) comprisingadministering (such as intravesicularly administering (for example viaurethral catheterization)) to the individual (a) an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin; (b) an effective amount of BCG; and (c) an effective amount ofmitomycin. In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin; (b) an effective amount of BCG; and (c) an effective amount ofmitomycin. In some embodiments, there is provided a method of treatingbladder cancer in an individual (e.g., human) comprising administering(such as intravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm); (b) aneffective amount of BCG; and (c) an effective amount of mitomycin. Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, and whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm); (b) an effective amountof BCG; and (c) an effective amount of mitomycin. In some embodiments,there is provided a method of treating bladder cancer in an individual(e.g., human) comprising administering (such as intravesicularlyadministering (for example via urethral catheterization)) to theindividual (a) an effective amount of a composition comprisingnanoparticles comprising sirolimus and human albumin, wherein thenanoparticles comprise sirolimus coated with the human albumin, whereinthe nanoparticles have an average particle size of no greater than about150 nm (such as no greater than about 120 nm, for example about 100 nm),wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1); (b)an effective amount of BCG; and (c) an effective amount of mitomycin. Insome embodiments, there is provided a method of treating bladder cancerin an individual (e.g., human) comprising administering (such asintravesicularly administering (for example via urethralcatheterization)) to the individual (a) an effective amount of acomposition comprising Nab-sirolimus; (b) an effective amount of BCG;and (c) an effective amount of mitomycin. In some embodiments, there isprovided a method of treating bladder cancer in an individual (e.g.,human) comprising administering (such as intravesicularly administering(for example via urethral catheterization)) administering to theindividual (a) an effective amount of Nab-sirolimus; (b) an effectiveamount of BCG; and (c) an effective amount of mitomycin. In someembodiments, the bladder cancer is non-muscle invasive bladder cancer.In some embodiments, the bladder cancer is BCG-refractory bladdercancer. In some embodiments, the bladder cancer is BCG-refractorynon-muscle invasive bladder cancer.

Use of Biomarkers

The present application further provides methods of treatments (such asany of the treatment methods described above) based on the expression ofone or more biomarkers. Biomarkers useful for methods described hereininclude, but are not limited to, p-S6K, pAKT, p-4EBP1, Ki67, p53, p63,Stathmin, Tau, SPARC, p73, c-myc, and cyclin D1. In some embodiments,the biomarker is selected from the group consisting of p-S6K, pAKT,p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc, TSC1, andcyclin D1.

PS6k encodes a member of the RSK (ribosomal s6 kinase) family ofserine/threonine kinases. This kinase contains 2 non-identical kinasecatalytic domains and phosphorylates several residues of the S6ribosomal protein. The kinase activity of this protein leads to anincrease in protein synthesis and cell proliferation.

Akt, also known as Protein Kinase B (PKB), is aserine/threonine-specific protein kinase that plays a key role inmultiple cellular processes, including, e.g., glucose metabolism,apoptosis, cell proliferation, transcription and cell migration.

p4EBP1 is a member of a family of translation repressor proteins. p4EBP1directly interacts with eukaryotic translation initiation factor 4E(eIF4E), which is a limiting component of the multi-subunit complex thatrecruits 40S ribosomal subunits to the 5′ end of mRNAs. Interaction ofthis protein with eIF4E inhibits complex assembly and repressestranslation. It has been shown that mTOR signals downstream to at leastS6K1 and 4EBP1/eIF4E, which themselves function in translational controlto regulate mammalian cell size (Fingar et al. (2002) Genes Dev. 16:1472-1487).

The Ki-67 protein (also known as MKI67) is a cellular marker forproliferation (Wu et al. (2003) Dev. Cell 5: 723-34). During interphase,Ki-67 can be exclusively detected within the cell nucleus, whereas inmitosis most of the protein is relocated to the surface of thechromosomes. Ki-67 is present during all active phases of the cell cycle(G1, S, G2, and mitosis), but is absent from resting cells (G0).

p53 (also known as protein 53 or tumor protein 53), is a tumorsuppressor protein that in humans is encoded by the TP53 gene. p53 is atranscription factor that is crucial in multicellular organisms, whereit regulates the cell cycle and, thus, functions as a tumor suppressorthat is involved in preventing cancer. p63 (also known as TP63) is amember of the p53 family of transcription factors. p63 −/− mice haveseveral developmental defects which include the lack of limbs and othertissues, such as teeth and mammary glands, which develop as a result ofinteractions between mesenchyme and epithelium. In humans, mutations inthe TP63 gene are associated with ectrodactyly-ectodermaldysplasia-cleft syndrome, Hay-Wells syndrome, cleft lip/palate syndrome3 (EEC3); ectrodactyly (also known as split-hand/foot malformation 4(SHFM4)); ankyloblepharon-ectodermal defects-cleft lip/palate, ADULTsyndrome (acro-dermato-ungual-lacrimal-tooth), limb-mammary syndrome,Rap-Hodgkin syndrome (RHS), and orofacial cleft 8. p73 (or TP73) wasfirst identified as a homologue of p53. The protein product of p73induces cell cycle arrest or apoptosis. Accordingly, p73 is classifiedas a tumor suppressor. However unlike p53, p73 is infrequently mutatedin cancers.

Stathmin 1/oncoprotein 18, also known as STMN1, is a highly conserved 17kDa protein that regulates microtubule dynamics. Stathmin forms acomplex with dimeric α,β-tubulin to form a ternary complex called theT2S complex. When stathmin sequesters tubulin into the T2S complex,tubulin becomes non-polymerizable. As a result, microtubule assembly isinhibited. Through this mechanism, stathmin promotes microtubuledisassembly.

Tau proteins are highly soluble microtubule-associated proteins (MAPs).In humans, these proteins are mostly found in neurons compared tonon-neuronal cells. For example, tau proteins are expressed in centralnervous system astrocytes and oligodendrocytes. One of tau's mainfunctions is to modulate the stability of axonal microtubules andpromote tubulin assembly into microtubules. Six tau isoforms exist inhuman brain tissue.

The Myc (c-Myc) gene encodes a transcription factor that activatesexpression of many genes through binding on Enhancer Box sequences(E-boxes) and recruiting histone acetyltransferases (HATs). Accordingly,Myc also functions to regulate global chromatin structure. Myc isactivated upon various mitogenic signals such as Wnt, Shh and EGF (viathe MAPK/ERK pathway). By modifying the expression of its target genes,Myc activation results in numerous biological effects. Myc plays rolesin driving cell proliferation, regulating cell growth and apoptosis, andregulating differentiation and stem cell self-renewal.

Cyclin D1 encodes the regulatory subunit of a holoenzyme thatphosphorylates and inactivates the retinoblastoma protein and promotesprogression through the G1-S phase of the cell cycle. Amplification oroverexpression of cyclin D1 plays pivotal roles in the development of asubset of human cancers including parathyroid adenoma, breast cancer,colon cancer, lymphoma, melanoma, and prostate cancer. Of the threeD-type cyclins, each of which binds cyclin-dependent kinase (CDK), it iscyclin D1 overexpression that is predominantly associated with humantumorigenesis and cellular metastases.

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellularprotein upregulated in several aggressive cancers. See Porter et al., J.Histochem. Cytochem. 1995; 43:791. The human SPARC gene encodes a 303amino acid SPARC proteins, while mature SPARC is a 285 amino acidglycoprotein. After cleavage of the signal sequence a 32-kD secretedform is produced which migrates at 43 kD on SDA-PAGE because ofglycosylation.

TSC1 (also referred to as Hamartin or tuberous sclerosis 1) is aperipheral membrane protein implicated as a tumor suppressor. It forms acomplex with TSC2 that regulates mTORC1 signaling and may be alsoinvolved in vesicular transport and docking.

Thus, the present invention in some embodiments provides a method oftreating bladder cancer in an individual (such as human) comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug), wherein the individual is selected for treatment based on thelevel of one or more of: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin,Tau, SPARC, p73, c-myc, and cyclin D1. In some embodiments, theindividual is selected for treatment based on the level of one of moreof: p-S6K, pAKT, p-4EBP1, and Ki67. In some embodiments, there isprovided a method of treating bladder cancer in an individual (such ashuman) comprising administering to the individual an effective amount ofa composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug), wherein the individual is selected for treatmentbased on the level of one or more of: p-S6K, pAKT, p-4EBP1, Ki67, p53,p63, Stathmin, Tau, SPARC, p73, c-myc, TSC1, and cyclin D1. In someembodiments, the individual is selected for treatment based on the levelof TSC1. In some embodiments, the method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin. In some embodiments, the methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the limus drug in the nanoparticles is coated with thealbumin. In some embodiments, the method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticles havean average particle size of no greater than about 200 nm (such as nogreater than about 150 nm). In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles comprise a limus drug coated with albumin, wherein thenanoparticles have an average particle size of no greater than about 200nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus. Insome embodiments, the method further comprises administering to theindividual an effective amount of another agent (such as BCG and/ormitomycin).

As used herein, “based upon” or “based on” include assessing,determining, or measuring the individual's characteristics as describedherein (and preferably selecting an individual suitable for receivingtreatment). When a biomarker is used as a basis for selection, assessing(or aiding in assessing), measuring, or determining method of treatmentas described herein, the biomarker is measured before and/or duringtreatment, and the values obtained are used by a clinician in assessingany of the following: (a) probable or likely suitability of anindividual to initially receive treatment(s); (b) probable or likelyunsuitability of an individual to initially receive treatment(s); (c)responsiveness to treatment; (d) probable or likely suitability of anindividual to continue to receive treatment(s); (e) probable or likelyunsuitability of an individual to continue to receive treatment(s); (f)adjusting dosage; or (g) predicting likelihood of clinical benefits.

Thus, the present invention in some embodiments provides a method oftreating bladder cancer in an individual (such as human) comprisingadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug), wherein the individual is selected for treatment based on a highlevel of one or more of: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin,Tau, SPARC, p73, c-myc, and cyclin D1. In some embodiments, theindividual is selected for treatment based on a high level of one ofmore of: p-S6K, pAKT, p-4EBP1, and Ki67. In some embodiments, the methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, the method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the limus drug in thenanoparticles is coated with the albumin. In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, the method comprises administering to the individualan effective amount of a composition comprising nanoparticles comprisinga limus drug and an albumin, wherein the nanoparticles comprise a limusdrug coated with albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, the method comprises administeringto the individual an effective amount of a composition comprisingnanoparticles comprising sirolimus and human albumin, wherein thenanoparticles comprise sirolimus coated with human albumin, wherein thenanoparticles have an average particle size of no greater than about 150nm (such as no greater than about 120 nm, for example about 100 nm),wherein the weight ratio of human albumin and sirolimus in thecomposition is about 9:1 or less (such as about 9:1 or about 8:1). Insome embodiments, the composition comprises Nab-sirolimus. In someembodiments, the composition is Nab-sirolimus. In some embodiments, thecomposition is Nab-sirolimus. In some embodiments, the method furthercomprises administering to the individual an effective amount of anotheragent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating bladdercancer in an individual (such as human) comprising administering to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug), wherein theindividual has a high level of one or more of: p-S6K, pAKT, p-4EBP1,Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc, and cyclin D1. In someembodiments, the individual has a high level of one of more of: p-S6K,pAKT, p-4EBP1, and Ki67. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug in the nanoparticlesis coated with the albumin. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles comprise a limus drug coated withalbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, the method comprises administering to the individualan effective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with human albumin, wherein the nanoparticles have anaverage particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1). In some embodiments, thecomposition comprises Nab-sirolimus. In some embodiments, thecomposition is Nab-sirolimus. In some embodiments, the composition isNab-sirolimus. In some embodiments, the method further comprisesadministering to the individual an effective amount of another agent(such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating bladdercancer in an individual (such as human) comprising: a) determining thelevel of one or more biomarkers selected from the group consisting ofp-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc,and cyclin D1 in the individual; and b) selecting the individual fortreatment based on the individual having a high level of one of more ofthe biomarkers, wherein the treatment method comprises administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a limus drug). Insome embodiments, the individual is selected for treatment based on theindividual having a high level of one of more biomarkers selected fromthe group consisting of p-S6K, pAKT, p-4EBP1, and Ki67. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, the method comprises administeringto the individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin, wherein thenanoparticles comprise a limus drug coated with albumin, wherein thenanoparticles have an average particle size of no greater than about 200nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus. Insome embodiments, the composition is Nab-sirolimus. In some embodiments,the method further comprises administering to the individual aneffective amount of another agent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating bladdercancer in an individual (such as human) comprising: a) selecting theindividual for treatment based on the individual having a high level ofone of more of the biomarkers selected from the group consisting ofp-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc,and cyclin D1 in the individual; b) administering to the selectedindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug). In someembodiments, the individual is selected for treatment based on theindividual having a high level of one of more biomarkers selected fromthe group consisting of p-S6K, pAKT, p-4EBP1, and Ki67. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the nanoparticles have an averageparticle size of no greater than about 200 nm (such as no greater thanabout 150 nm). In some embodiments, the method comprises administeringto the individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin, wherein thenanoparticles comprise a limus drug coated with albumin, wherein thenanoparticles have an average particle size of no greater than about 200nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus. Insome embodiments, the composition is Nab-sirolimus. In some embodiments,the method further comprises administering to the individual aneffective amount of another agent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating bladdercancer in an individual (such as human) comprising: a) determining thelevel of one or more biomarkers selected from the group consisting ofp-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc,and cyclin D1 in the individual; and b) selecting the individual fortreatment based on the individual having a high level of one of more ofthe biomarkers, c) administering to the selected individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor (such as a limus drug). In some embodiments, the individual isselected for treatment based on the individual having a high level ofone of more biomarkers selected from the group consisting of p-S6K,pAKT, p-4EBP1, and Ki67. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the method comprises administering to the individual aneffective amount of a composition comprising nanoparticles comprising alimus drug and an albumin, wherein the limus drug in the nanoparticlesis coated with the albumin. In some embodiments, the method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments, themethod comprises administering to the individual an effective amount ofa composition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles comprise a limus drug coated withalbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, the method comprises administering to the individualan effective amount of a composition comprising nanoparticles comprisingsirolimus and human albumin, wherein the nanoparticles comprisesirolimus coated with human albumin, wherein the nanoparticles have anaverage particle size of no greater than about 150 nm (such as nogreater than about 120 nm, for example about 100 nm), wherein the weightratio of human albumin and sirolimus in the composition is about 9:1 orless (such as about 9:1 or about 8:1). In some embodiments, thecomposition comprises Nab-sirolimus. In some embodiments, thecomposition is Nab-sirolimus. In some embodiments, the composition isNab-sirolimus. In some embodiments, the method further comprisesadministering to the individual an effective amount of another agent(such as BCG and/or mitomycin).

In some embodiments, there is provided a method of assessing whether anindividual with bladder cancer is more likely to respond to a treatmentcomprising administering to the selected individual an effective amountof a composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug), the method comprising assessing the level of oneor more biomarkers selected from the group consisting of p-S6K, pAKT,p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc, and cyclin D1in the individual; wherein a low level of one or more of the biomarkersindicates that the individual is less likely to respond to thetreatment, wherein a high level of one of more of the biomarkersindicates that the individual is more likely to respond to thetreatment. In some embodiments, the method further comprises selectingthe individual for treatment based on the individual having a high levelof one of more of the biomarkers. In some embodiments, the methodfurther comprises administering to the selected individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor (such as a limus drug). In some embodiments, the individual isselected for treatment based on the individual having a high level ofone of more biomarkers selected from the group consisting of p-S6K,pAKT, p-4EBP1, and Ki67. In some embodiments, the treatment methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin. In some embodiments, the treatment method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe limus drug in the nanoparticles is coated with the albumin. In someembodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticles havean average particle size of no greater than about 200 nm (such as nogreater than about 150 nm). In some embodiments, the treatment methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising a limus drug and analbumin, wherein the nanoparticles comprise a limus drug coated withalbumin, wherein the nanoparticles have an average particle size of nogreater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising sirolimus and human albumin, wherein the nanoparticlescomprise sirolimus coated with human albumin, wherein the nanoparticleshave an average particle size of no greater than about 150 nm (such asno greater than about 120 nm, for example about 100 nm), wherein theweight ratio of human albumin and sirolimus in the composition is about9:1 or less (such as about 9:1 or about 8:1). In some embodiments, thecomposition comprises Nab-sirolimus. In some embodiments, thecomposition is Nab-sirolimus. In some embodiments, the composition isNab-sirolimus. In some embodiments, the treatment method furthercomprises administering to the individual an effective amount of anotheragent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of determining whetheran individual with bladder cancer has responded to a treatmentcomprising administering to the selected individual an effective amountof a composition comprising nanoparticles comprising an mTOR inhibitor(such as a limus drug), the method comprising assessing the level of oneor more biomarkers selected from the group consisting of p-S6K, pAKT,p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc, and cyclin D1in the individual prior to and after the treatment; wherein a decreasedlevel of one or more of the biomarkers after the treatment indicatesthat the individual has responded to the treatment. In some embodiments,the method further comprises continue to administer to the individualwho has responded to the treatment an effective amount of a compositioncomprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug). In some embodiments, the method comprises adjusting the dosage ofthe mTOR nanoparticle composition. In some embodiments, the biomarker isselected from the group consisting of p-S6K, pAKT, p-4EBP1, and Ki67. Insome embodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin. In some embodiments, thetreatment method comprises administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the limus drug in the nanoparticles is coatedwith the albumin. In some embodiments, the treatment method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin, whereinthe nanoparticles have an average particle size of no greater than about200 nm (such as no greater than about 150 nm). In some embodiments, thetreatment method comprises administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles comprise a limus drug coatedwith albumin, wherein the nanoparticles have an average particle size ofno greater than about 200 nm (such as no greater than about 150 nm). Insome embodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising sirolimus and human albumin, wherein the nanoparticlescomprise sirolimus coated with human albumin, wherein the nanoparticleshave an average particle size of no greater than about 150 nm (such asno greater than about 120 nm, for example about 100 nm), wherein theweight ratio of human albumin and sirolimus in the composition is about9:1 or less (such as about 9:1 or about 8:1). In some embodiments, thecomposition comprises Nab-sirolimus. In some embodiments, thecomposition is Nab-sirolimus. In some embodiments, the composition isNab-sirolimus. In some embodiments, the treatment method furthercomprises administering to the individual an effective amount of anotheragent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating anindividual having bladder cancer, comprising (i) administering to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug), (ii) assessing thelevel of one or more biomarkers selected from the group consisting ofp-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc,and cyclin D1 in the individual after the treatment; (iii) comparing thelevels of the one or more biomarkers with the level of the biomarkersprior to the treatment, and (iv) continue to administer to theindividual an effective amount of a composition comprising nanoparticlescomprising an mTOR inhibitor (such as a limus drug) if the individualhas a decreased level of one or more of the biomarkers after thetreatment. In some embodiments, the method comprises adjusting thedosage of the mTOR nanoparticle composition. In some embodiments, thebiomarker is selected from the group consisting of p-S6K, pAKT, p-4EBP1,and Ki67. In some embodiments, the treatment method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the limus drug in thenanoparticles is coated with the albumin. In some embodiments, thetreatment method comprises administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles have an average particle sizeof no greater than about 200 nm (such as no greater than about 150 nm).In some embodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm). In some embodiments, the treatment methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus. Insome embodiments, the composition is Nab-sirolimus. In some embodiments,the treatment method further comprises administering to the individualan effective amount of another agent (such as BCG and/or mitomycin).

In some embodiments, there is provided a method of treating anindividual having bladder cancer, comprising: (i) assessing the level ofone or more biomarkers selected from the group consisting of p-S6K,pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73, c-myc, andcyclin D1 in the individual prior to treatment; (ii) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a limus drug), (iii)assessing the level of one or more biomarkers selected from the groupconsisting of p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau,SPARC, p73, c-myc, and cyclin D1 in the individual after the treatment;(iv) comparing the levels of the one or more biomarkers with the levelof the biomarkers prior to the treatment, and (v) continue to administerto the individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (such as a limus drug) if theindividual has a decreased level of one or more of the biomarkers afterthe treatment. In some embodiments, the method comprises adjusting thedosage of the mTOR nanoparticle composition. In some embodiments, thebiomarker is selected from the group consisting of p-S6K, pAKT, p-4EBP1,and Ki67. In some embodiments, the treatment method comprisesadministering to the individual an effective amount of a compositioncomprising nanoparticles comprising a limus drug and an albumin. In someembodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the limus drug in thenanoparticles is coated with the albumin. In some embodiments, thetreatment method comprises administering to the individual an effectiveamount of a composition comprising nanoparticles comprising a limus drugand an albumin, wherein the nanoparticles have an average particle sizeof no greater than about 200 nm (such as no greater than about 150 nm).In some embodiments, the treatment method comprises administering to theindividual an effective amount of a composition comprising nanoparticlescomprising a limus drug and an albumin, wherein the nanoparticlescomprise a limus drug coated with albumin, wherein the nanoparticleshave an average particle size of no greater than about 200 nm (such asno greater than about 150 nm). In some embodiments, the treatment methodcomprises administering to the individual an effective amount of acomposition comprising nanoparticles comprising sirolimus and humanalbumin, wherein the nanoparticles comprise sirolimus coated with humanalbumin, wherein the nanoparticles have an average particle size of nogreater than about 150 nm (such as no greater than about 120 nm, forexample about 100 nm), wherein the weight ratio of human albumin andsirolimus in the composition is about 9:1 or less (such as about 9:1 orabout 8:1). In some embodiments, the composition comprisesNab-sirolimus. In some embodiments, the composition is Nab-sirolimus. Insome embodiments, the composition is Nab-sirolimus. In some embodiments,the treatment method further comprises administering to the individualan effective amount of another agent (such as BCG and/or mitomycin).

The methods described herein in some embodiments comprise determiningthe levels of one or more biomarkers in an individual. In someembodiments, the level is the activity level of a biomarker in a sample.In some embodiments the level is an expression level. In someembodiments the level is a measure of a protein present in a cell (forexample the surface of a cell), a sample, or a tumor. In someembodiments, the level is based on a mutation or polymorphism in thebiomarker gene that correlates with the protein or mRNA level of abiomarker. In some embodiments, the level is the protein expressionlevel. In some embodiments, the level is the mRNA level.

Levels of biomarker in an individual may be determined based on a sample(e.g., sample from the individual or reference sample). In someembodiments, the sample is from a tissue, organ, cell, or tumor. In someembodiments, the sample is a biological sample. In some embodiments, thebiological sample is a biological fluid sample or a biological tissuesample. In further embodiments, the biological fluid sample is a bodilyfluid. Bodily fluids include, but are not limited to, blood, urine,lymph, saliva, semen, peritoneal fluid, cerebrospinal fluid, breastmilk, and pleural effusion. In some embodiments, the sample is a bloodsample which includes, for example, platelets, lymphocytes,polymorphonuclear cells, macrophages, and erythrocytes. In someembodiments, the sample is a urine sample.

In some embodiments, the sample is a tumor tissue, normal tissueadjacent to said tumor, normal tissue distal to said tumor, bloodsample, or other biological sample. In some embodiments, the sample is afixed sample. Fixed samples include, but are not limited to, a formalinfixed sample, a paraffin-embedded sample, or a frozen sample. In someembodiments, the sample is a biopsy containing cancer cells. In someembodiments, the sample is a biopsy sample obtained from a cystoscopy.In some embodiments, the sample is a biopsy sample obtained from a transurethral resection of the bladder tumor (TURBT). In a furtherembodiment, the biopsy is a fine needle aspiration of bladder cancercells. In a further embodiment, the biopsy is laparoscopy obtainedbladder cancer cells. In some embodiments, the biopsied cells arecentrifuged into a pellet, fixed, and embedded in paraffin. In someembodiments, the biopsied cells are flash frozen. In some embodiments,the biopsied cells are mixed with an antibody that recognizes thebiomarker. In some embodiments, a biopsy is taken to determine whetheran individual has cancer and is then used as a sample. In someembodiments, the sample comprises surgically obtained tumor cells. Insome embodiments, samples may be obtained at different times than whenthe determining of biomarker levels occurs.

In some embodiments, the sample comprises a circulating metastaticbladder cancer cell. In some embodiments, the sample is obtained bysorting bladder circulating tumor cells (CTCs) from blood. In a furtherembodiment, the CTCs have detached from a primary tumor and circulate ina bodily fluid. In yet a further embodiment, the CTCs have detached froma primary tumor and circulate in the bloodstream. In a furtherembodiment, the CTCs are an indication of metastasis.

In some embodiments, the level of one biomarker (such as p-S6K) isdetermined. In some embodiments, the levels of two or more biomarkersare determined; for example, one or more biomarkers selected from thegroup consisting of p-S6K, pAKT, p-4EBP1, and Ki67 can be determined.The one or more biomarkers include, for example, at least two or morebiomarkers, at least three or more biomarkers, at least four or morebiomarkers, at least five or more biomarkers, or at least six or morebiomarkers described herein. In some embodiments, the one or morebiomarkers include p-S6K.

In some embodiments, the protein expression level of the biomarker isdetermined. In some embodiments, the mRNA level of the biomarker isdetermined. In some embodiments, the level of the biomarker isdetermined by an immunohistochemistry method.

The levels of a biomarker may be a high level or a low level as comparedto a control sample. In some embodiments, the level of the biomarker inan individual is compared to the level of the biomarker in a controlsample. In some embodiments the level of the biomarker in a subject iscompared to the level of the biomarker in multiple control samples. Insome embodiments, multiple control samples are used to generate astatistic that is used to classify the level of the biomarker in anindividual with cancer.

In some embodiments, the DNA copy number is determined, and a high DNAcopy number for the gene encoding the biomarker (for example a high DNAcopy number as compared to a control sample) is indicative of a highlevel of the biomarker.

The classification or ranking of the biomarker level (i.e., high or low)may be determined relative to a statistical distribution of controllevels. In some embodiments, the classification or ranking is relativeto a control sample obtained from the individual. In some embodiment thelevels of the biomarker (such as p-S6K) is classified or ranked relativeto a statistical distribution of control levels. In some embodiments,the level of the biomarker (such as p-S6K1) is classified or rankedrelative to the level from a control sample obtained from the subject.

Control samples can be obtained using the same sources and methods asnon-control samples. In some embodiments, the control sample is obtainedfrom a different individual (for example an individual not having cancerand/or an individual sharing similar ethnic, age, and gender identity).In some embodiments when the sample is a tumor tissue sample, thecontrol sample may be a non-cancerous sample from the same individual.In some embodiments, multiple control samples (for example fromdifferent individuals) are used to determine a range of levels ofbiomarkers in a particular tissue, organ, or cell population. In someembodiments, the control sample is a cultured tissue or cell that hasbeen determined to be a proper control. In some embodiments, the controlis a cell that does not express the biomarker. In some embodiments, aclinically accepted normal level in a standardized test is used as acontrol level for determining the biomarker level. In some embodiments,the reference level of biomarker (e.g., p-S6K1) in the subject isclassified as high, medium or low according to a scoring system, such asan immunohistochemistry-based scoring system. In some embodiments, thereference level of biomarker (e.g., p-S6K1) in the subject is classifiedas a low sample when the score is less than or equal to the overallmedian score.

In some embodiments, the biomarker level is determined by measuring thelevel of a biomarker in an individual and comparing to a control orreference (e.g., the median level for the given patient population orlevel of a second individual). For example, if the level of a biomarker(e.g., p-S6K1) for the single individual is determined to be above themedian level of the patient population, that individual is determined tohave a high level of the biomarker. Alternatively, if the level of abiomarker for the single individual is determined to be below the medianlevel of the patient population, that individual is determined to have alow level of the biomarker. In some embodiments, the individual iscompared to a second individual and/or a patient population which isresponsive to treatment. In some embodiments, the individual is comparedto a second individual and/or a patient population which is notresponsive to treatment. In any of the embodiments herein, the levelscan be determined by measuring the level of a nucleic acid encoding abiomarker (e.g., p-S6K1). For example, if the level of an mRNA encodinga biomarker for the single individual is determined to be above themedian level of the patient population, that individual is determined tohave a high level of an mRNA encoding the biomarker. Alternatively, ifthe level of mRNA encoding the biomarker for the single individual isdetermined to be below the median level of the patient population, thatindividual is determined to have a low level of an mRNA encoding thebiomarker.

In some embodiments, the reference level of a biomarker is determined byobtaining a statistical distribution of biomarker levels.

In some embodiments, bioinformatics methods are used for thedetermination and classification of the levels of the biomarker.Numerous alternative bioinformatics approaches have been developed toassess gene set expression profiles using gene expression profilingdata. Methods include but are not limited to those described in Segal,E. et al. Nat. Genet. 34:66-176 (2003); Segal, E. et al. Nat. Genet.36:1090-1098 (2004); Barry, W. T. et al. Bioinformatics 21:1943-1949(2005); Tian, L. et al. Proc Nat'l Acad Sci USA 102:13544-13549 (2005);Novak B A and Jain A N. Bioinformatics 22:233-41 (2006); Maglietta R etal. Bioinformatics 23:2063-72 (2007); Bussemaker H J, BMC Bioinformatics8 Suppl 6:S6 (2007).

In some embodiments, mRNA level is determined, and a low level is anmRNA level less than about 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3,5, 7, 10, 20, 50, 70, 100, 200, 500, 1000 times or less than 1000 timesto that of what is considered as clinically normal or to the levelobtained from a control. In some embodiments, high level is an mRNAlevel more than about 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3, 5,7, 10, 20, 50, 70, 100, 200, 500, 1000 times or more than 1000 times tothat of what is considered as clinically normal or to the level obtainedfrom a control.

In some embodiments, protein expression level is determined, for exampleby immunohistochemistry. For example, the criteria for low or highlevels can be made based on the number of positive staining cells and/orthe intensity of the staining, for example by using an antibody thatspecifically recognizes the biomarker protein (e.g., p-S6K). In someembodiments, the level is low if less than about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or 50% cells have positive staining. In someembodiments, the level is low if the staining is 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or 50% less intense than a positive controlstaining.

In some embodiments, the level is high if more than about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, cells have positive staining.In some embodiments, the level is high if the staining is as intense aspositive control staining. In some embodiments, the level is high if thestaining is 80%, 85%, or 90% as intense as positive control staining.

In some embodiments, strong staining, moderate staining, and weakstaining are calibrated levels of staining, wherein a range isestablished and the intensity of staining is binned within the range. Insome embodiments, strong staining is staining above the 75th percentileof the intensity range, moderate staining is staining from the 25th tothe 75th percentile of the intensity range, and low staining is stainingis staining below the 25th percentile of the intensity range. In someaspects one skilled in the art, and familiar with a particular stainingtechnique, adjusts the bin size and defines the staining categories.

Further provided herein are methods of directing treatment of a bladdercancer by delivering a sample to a diagnostic lab for determination ofbiomarker levels; providing a control sample with a known level of abiomarker; providing an antibody to a biomarker (e.g., p-S6K1 antibody);subjecting the sample and control sample to binding by the antibody,and/or detecting a relative amount of antibody binding, wherein thelevel of the sample is used to provide a conclusion that a patientshould receive a treatment with any one of the methods described herein.

Also provided herein are methods of directing treatment of a disease,further comprising reviewing or analyzing data relating to the presence(or level) of a biomarker (e.g., p-S6K1) in a sample; and providing aconclusion to an individual about the likelihood or suitability of theindividual to respond to a treatment, a health care provider or a healthcare manager, the conclusion being based on the review or analysis ofdata. In one aspect of the invention a conclusion is the transmission ofthe data over a network.

Dosing and Method of Administering the Nanoparticle Compositions

The dose of the mTOR nanoparticles (such as a limus nanoparticlecompositions) administered to an individual (such as a human) may varywith the particular composition, the mode of administration, and thetype of bladder cancer being treated. In some embodiments, the amount ofthe composition is effective to result in an objective response (such asa partial response or a complete response). In some embodiments, theamount of the mTOR nanoparticle composition (such as a limusnanoparticle composition) is sufficient to result in a complete responsein the individual. In some embodiments, the amount of the mTORnanoparticle composition (such as a limus nanoparticle composition) issufficient to result in a partial response in the individual. In someembodiments, the amount of the mTOR nanoparticle composition (such as alimus nanoparticle composition) administered (for example whenadministered alone) is sufficient to produce an overall response rate ofmore than about any of 20%, 30%, 40%, 50%, 60%, or 64% among apopulation of individuals treated with the mTOR nanoparticle composition(such as a limus nanoparticle composition). Responses of an individualto the treatment of the methods described herein can be determined, forexample, based on RECIST levels, cystoscopy (with or without biopsy),biopsy, cytology, and CT imaging.

In some embodiments, the amount of the mTOR nanoparticle composition(such as a limus nanoparticle composition) is sufficient to produce anegative biopsy in the individual. In some embodiments, the amount ofthe mTOR nanoparticle composition (such as a limus nanoparticlecomposition) is sufficient to produce a response (partial or complete)based on urine cytology. In some embodiments, the amount of the mTORnanoparticle composition (such as a limus nanoparticle composition) issufficient to produce both a negative biopsy and a response (partial orcomplete) based on urine cytology.

In some embodiments, the amount of the mTOR nanoparticle composition(such as a limus nanoparticle composition) is not sufficient to causesystemic toxicity, such as cystitis, hematuria, dysuria, urinaryretension, urinary frequency/urgency, or bladder spasm.

In some embodiments, the amount of the composition is sufficient toprolong progress-free survival of the individual. In some embodiments,the amount of the composition is sufficient to prolong overall survivalof the individual. In some embodiments, the amount of the composition(for example when administered alone) is sufficient to produce clinicalbenefit of more than about any of 50%, 60%, 70%, or 77% among apopulation of individuals treated with the mTOR nanoparticle composition(such as a limus nanoparticle composition).

In some embodiments, the amount of the composition is an amountsufficient to decrease the size of a tumor, decrease the number ofcancer cells, or decrease the growth rate of a tumor by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% comparedto the corresponding tumor size, number of bladder cancer cells, ortumor growth rate in the same subject prior to treatment or compared tothe corresponding activity in other subjects not receiving thetreatment. Standard methods can be used to measure the magnitude of thiseffect, such as in vitro assays with purified enzyme, cell-based assays,animal models, or human testing.

In some embodiments, the amount of the mTOR inhibitor (such as a limusdrug, for example sirolimus) in the composition is below the level thatinduces a toxicological effect (i.e., an effect above a clinicallyacceptable level of toxicity) or is at a level where a potential sideeffect can be controlled or tolerated when the composition isadministered to the individual.

In some embodiments, the amount of the composition is close to a maximumtolerated dose (MTD) of the composition following the same dosingregime. In some embodiments, the amount of the composition is more thanabout any of 80%, 90%, 95%, or 98% of the MTD.

In some embodiments, the amount of an mTOR inhibitor (such as a limusdrug, e.g., sirolimus) in the composition is included in any of thefollowing ranges: about 0.1 mg to about 1000 mg, about 0.1 mg to about2.5 mg, about 0.5 mg to about 5 mg, about 5 mg to about 10 mg, about 10mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg to about 25mg, about 20 mg to about 50 mg, about 25 mg to about 50 mg, about 50 mgto about 75 mg, about 50 mg to about 100 mg, about 75 mg to about 100mg, about 100 mg to about 125 mg, about 125 mg to about 150 mg, about150 mg to about 175 mg, about 175 mg to about 200 mg, about 200 mg toabout 225 mg, about 225 mg to about 250 mg, about 250 mg to about 300mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about400 mg to about 450 mg, or about 450 mg to about 500 mg, about 500 mg toabout 600 mg, about 600 mg to about 700 mg, about 700 mg to about 800mg, about 800 mg to about 900 mg, or about 900 mg to about 1000 mg. Insome embodiments, the amount of an mTOR inhibitor (such as a limus drug,e.g., sirolimus) in the effective amount of the composition (e.g., aunit dosage form) is in the range of about 5 mg to about 500 mg, such asabout 30 mg to about 400 mg, 30 mg to about 300 mg, or about 50 mg toabout 200 mg. In some embodiments, the amount of an mTOR inhibitor (suchas a limus drug, e.g., sirolimus) in the effective amount of thecomposition (e.g., a unit dosage form) is in the range of about 150 mgto about 500 mg, including for example, about 150 mg, about 225 mg,about 250 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg,about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about 500 mg.In some embodiments, the concentration of the mTOR inhibitor (such as alimus drug, e.g., sirolimus) in the composition is dilute (about 0.1mg/ml) or concentrated (about 100 mg/ml), including for example any ofabout 0.1 to about 50 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 1mg/ml to about 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 mg/mlto about 6 mg/ml, or about 5 mg/ml. In some embodiments, theconcentration of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) is at least about any of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml.

In some embodiments of any of the above aspects, the amount of an mTORinhibitor (such as a limus drug, e.g., sirolimus) in the compositionincludes at least about any of 1 mg/kg, 2.5 mg/kg, 3.5 mg/kg, 5 mg/kg,6.5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, or 60 mg/kg. Invarious embodiments, the effective amount of an mTOR inhibitor (such asa limus drug, e.g., sirolimus) in the composition includes less thanabout any of 350 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150 mg/kg, 100mg/kg, 50 mg/kg, 25 mg/kg, 20 mg/kg, 10 mg/kg, 7.5 mg/kg, 6.5 mg/kg, 5mg/kg, 3.5 mg/kg, 2.5 mg/kg, or 1 mg/kg of an mTOR inhibitor (such as alimus drug, e.g., sirolimus).

Exemplary dosing frequencies for the administration of the nanoparticlecompositions include, but are not limited to, daily, every two days,every three days, every four days, every five days, every six days,weekly without break, three out of four weeks, once every three weeks,once every two weeks, or two out of three weeks. In some embodiments,the composition is administered about once every 2 weeks, once every 3weeks, once every 4 weeks, once every 6 weeks, or once every 8 weeks. Insome embodiments, the composition is administered at least about any of1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) a week. In some embodiments,the intervals between each administration are less than about any of 6months, 3 months, 1 month, 20 days, 15, days, 14 days, 13 days, 12 days,11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3days, 2 days, or 1 day. In some embodiments, the intervals between eachadministration are more than about any of 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 8 months, or 12 months. In some embodiments,there is no break in the dosing schedule. In some embodiments, theinterval between each administration is no more than about a week. Insome embodiments, the composition is administered weekly. In someembodiments, the composition is administered twice every week.

The administration of the composition can be extended over an extendedperiod of time, such as from about a month up to about seven years. Insome embodiments, the composition is administered over a period of atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36,48, 60, 72, or 84 months. In some embodiments, the composition isadministered weekly for 6 weeks, optionally followed by monthlymaintenance thereafter.

In some embodiments, the individual is treated for at least about any ofone, two, three, four, five, six, seven, eight, nine, or ten treatmentcycles.

The mTOR nanoparticle composition (such as a limus nanoparticlecomposition) can be administered to an individual (such as human) viavarious routes, including, for example, intravenous, intra-arterial,intraperitoneal, intrapulmonary, oral, inhalation, intravesicular,intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal,transmucosal, and transdermal. In some embodiments, sustained continuousrelease formulation of the composition may be used. In some embodiments,the composition is administered intravenously. In some embodiments, thecomposition is administered intravesicularly. In some embodiments, thecomposition is administered intraarterially. In some embodiments, thecomposition is administered intraperitoneally.

In some embodiments when the limus nanoparticle composition isadministered intravesicularly, the dosage of an mTOR inhibitor (such asa limus drug, e.g., sirolimus) in a nanoparticle composition can be inthe range of about 30 mg to about 400 mg in volume of about 20 ml toabout 150 ml, for example retained in the bladder for about 30 minutesto about 4 hours. In some embodiments, the nanoparticle composition isretained in the bladder for about 30 minutes to about 4 hours, includingfor example about 30 minutes to about 1 hour, about 1 hour to about 2hours, about 2 hours to about 3 hours, or about 3 hours to about 4hours.

In some embodiments, the dosage of an mTOR inhibitor (such as a limusdrug, e.g., sirolimus) is about 100 mg to about 400 mg, for exampleabout 100 mg, about 200 mg, about 300 mg, or about 400 mg. In someembodiments, the limus drug is administered at about 100 mg weekly,about 200 mg weekly, about 300 mg weekly, about 100 mg twice weekly, orabout 200 mg twice weekly. In some embodiments, the administration isfurther followed by a monthly maintenance dose (which can be the same ordifferent from the weekly doses).

In some embodiments when the limus nanoparticle composition isadministered intravenously, the dosage of an mTOR inhibitor (such as alimus drug, e.g., sirolimus) in a nanoparticle composition can be in therange of about 30 mg to about 400 mg. The compositions described hereinallow infusion of the composition to an individual over an infusion timethat is shorter than about 24 hours. For example, in some embodiments,the composition is administered over an infusion period of less thanabout any of 24 hours, 12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, thecomposition is administered over an infusion period of about 30 minutesto about 40 minutes.

Modes of Administration of Combination Therapies

The dosing regimens described in the section above apply to bothmonotherapy and combination therapy settings. The modes ofadministration for combination therapy methods are further describedbelow.

In some embodiments, the nanoparticle composition and the other agent(including the specific chemotherapeutic agents described herein) areadministered simultaneously. When the drugs are administeredsimultaneously, the drug in the nanoparticles and the other agent may becontained in the same composition (e.g., a composition comprising boththe nanoparticles and the other agent) or in separate compositions(e.g., the nanoparticles are contained in one composition and the otheragent is contained in another composition).

In some embodiments, the nanoparticle composition and the other agentare administered sequentially. Either the nanoparticle composition orthe other agent may be administered first. The nanoparticle compositionand the other agent are contained in separate compositions, which may becontained in the same or different packages.

In some embodiments, the administration of the nanoparticle compositionand the other agent are concurrent, i.e., the administration period ofthe nanoparticle composition and that of the other agent overlap witheach other. In some embodiments, the nanoparticle composition isadministered for at least one cycle (for example, at least any of 2, 3,or 4 cycles) prior to the administration of the other agent. In someembodiments, the other agent is administered for at least any of one,two, three, or four weeks. In some embodiments, the administrations ofthe nanoparticle composition and the other agent are initiated at aboutthe same time (for example, within any one of 1, 2, 3, 4, 5, 6, or 7days). In some embodiments, the administrations of the nanoparticlecomposition and the other agent are terminated at about the same time(for example, within any one of 1, 2, 3, 4, 5, 6, or 7 days). In someembodiments, the administration of the other agent continues (forexample for about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months) after the termination of the administration of the nanoparticlecomposition. In some embodiments, the administration of the other agentis initiated after (for example after about any one of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 months) the initiation of the administration ofthe nanoparticle composition. In some embodiments, the administrationsof the nanoparticle composition and the other agent are initiated andterminated at about the same time. In some embodiments, theadministrations of the nanoparticle composition and the other agent areinitiated at about the same time and the administration of the otheragent continues (for example for about any one of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 months) after the termination of the administrationof the nanoparticle composition. In some embodiments, the administrationof the nanoparticle composition and the other agent stop at about thesame time and the administration of the other agent is initiated after(for example after about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months) the initiation of the administration of the nanoparticlecomposition.

In some embodiments, the administration of the nanoparticle compositionand the other agent are non-concurrent. For example, in someembodiments, the administration of the nanoparticle composition isterminated before the other agent is administered. In some embodiments,the administration of the other agent is terminated before thenanoparticle composition is administered. The time period between thesetwo non-concurrent administrations can range from about two to eightweeks, such as about four weeks.

The dosing frequency of the drug-containing nanoparticle composition andthe other agent may be adjusted over the course of the treatment, basedon the judgment of the administering physician. When administeredseparately, the drug-containing nanoparticle composition and the otheragent can be administered at different dosing frequency or intervals.For example, the drug-containing nanoparticle composition can beadministered weekly, while a chemotherapeutic agent can be administeredmore or less frequently. In some embodiments, sustained continuousrelease formulation of the drug-containing nanoparticle and/orchemotherapeutic agent may be used. Various formulations and devices forachieving sustained release are known in the art. A combination of theadministration configurations described herein can also be used.

The nanoparticle composition and the other agent can be administeredusing the same route of administration or different routes ofadministration. In some embodiments (for both simultaneous andsequential administrations), the limus drug in the nanoparticlecomposition and the other agent are administered at a predeterminedratio. For example, in some embodiments, the ratio by weight of thelimus drug in the nanoparticle composition and the other agent is about1 to 1. In some embodiments, the weight ratio may be between about 0.001to about 1 and about 1000 to about 1, or between about 0.01 to about 1and 100 to about 1. In some embodiments, the ratio by weight of thelimus drug in the nanoparticle composition and the other agent is lessthan about any of 100:1, 50:1, 30:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, and 1:1 In some embodiments, the ratio by weight of the limusdrug in the nanoparticle composition and the other agent is more thanabout any of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 30:1, 50:1,100:1. Other ratios are contemplated.

The doses required for the limus drug and/or the other agent may (butnot necessarily) be lower than what is normally required when each agentis administered alone. Thus, in some embodiments, a subtherapeuticamount of the drug in the nanoparticle composition and/or the otheragent is administered. “Subtherapeutic amount” or “subtherapeutic level”refer to an amount that is less than the therapeutic amount, that is,less than the amount normally used when the drug in the nanoparticlecomposition and/or the other agent are administered alone. The reductionmay be reflected in terms of the amount administered at a givenadministration and/or the amount administered over a given period oftime (reduced frequency).

In some embodiments, enough chemotherapeutic agent is administered so asto allow reduction of the normal dose of the drug in the nanoparticlecomposition required to effect the same degree of treatment by at leastabout any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. Insome embodiments, enough drug in the nanoparticle composition isadministered so as to allow reduction of the normal dose of the otheragent required to effect the same degree of treatment by at least aboutany of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more.

In some embodiments, the dose of both the limus drug in the nanoparticlecomposition and the other agent are reduced as compared to thecorresponding normal dose of each when administered alone. In someembodiments, both the limus drug in the nanoparticle composition and theother agent are administered at a subtherapeutic, i.e., reduced, level.In some embodiments, the dose of the nanoparticle composition and/or theother agent is substantially less than the established maximum toxicdose (MTD). For example, the dose of the nanoparticle composition and/orthe other agent is less than about 50%, 40%, 30%, 20%, or 10% of theMTD.

A combination of the administration configurations described herein canbe used. The combination therapy methods described herein may beperformed alone or in conjunction with another therapy, such aschemotherapy, radiation therapy, surgery, hormone therapy, gene therapy,immunotherapy, chemoimmunotherapy, hepatic artery-based therapy,cryotherapy, ultrasound therapy, liver transplantation, local ablativetherapy, radiofrequency ablation therapy, photodynamic therapy, and thelike. Additionally, a person having a greater risk of developing thebladder cancer may receive treatments to inhibit and/or delay thedevelopment of the disease.

The other agent described herein can be administered to an individual(such as human) via various routes, such as parenterally, includingintravenous, intra-arterial, intraperitoneal, intrapulmonary, oral,inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal. In some embodiments, the otheragent is administrated intravenously. In some embodiments, thenanoparticle composition is administered orally.

The dosing frequency of the other agent can be the same or differentfrom that of the nanoparticle composition. Exemplary frequencies areprovided above. As further example, the other agent can be administeredthree times a day, two times a day, daily, 6 times a week, 5 times aweek, 4 times a week, 3 times a week, two times a week, weekly. In someembodiments, the other agent is administered twice daily or three timesdaily. Exemplary amounts of the other agent include, but are not limitedto, any of the following ranges: about 0.5 mg to about 5 mg, about 5 mgto about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg,about 20 mg to about 25 mg, about 20 mg to about 50 mg, about 25 mg toabout 50 mg, about 50 mg to about 75 mg, about 50 mg to about 100 mg,about 75 mg to about 100 mg, about 100 mg to about 125 mg, about 125 mgto about 150 mg, about 150 mg to about 175 mg, about 175 mg to about 200mg, about 200 mg to about 225 mg, about 225 mg to about 250 mg, about250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg toabout 400 mg, about 400 mg to about 450 mg, or about 450 mg to about 500mg. For example, the other agent can be administered at a dose of about1 mg/kg to about 200 mg/kg (including for example about 1 mg/kg to about20 mg/kg, about 20 mg/kg to about 40 mg/kg, about 40 mg/kg to about 60mg/kg, about 60 mg/kg to about 80 mg/kg, about 80 mg/kg to about 100mg/kg, about 100 mg/kg to about 120 mg/kg, about 120 mg/kg to about 140mg/kg, about 140 mg/kg to about 200 mg/kg).

In some embodiments the other agent is BCG. In some embodiments, thedose of BCG is about 1 mg to about 5 mg, about 5 mg to about 10 mg,about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg toabout 40 mg, about 40 mg to about 50 mg, about 50 mg to about 60 mg,about 60 mg to about 70 mg, about 70 mg to about 80 mg, about 80 mg toabout 90 mg. In some embodiments, the dose of BCG is about 1×10⁵ CFU/mlto about 1×10⁷ CFU/ml, including for example about 1-8×10⁶ CFU/ml, suchas about 2×10⁶, about 3×10⁶, about 4×10⁶, about 5×10⁶, about 6×10⁶,about 7×10⁶, or about 8×10⁶ CFU/ml. In some embodiments, the BCG isadministered intravesicularly. In some embodiments, the BCG isadministered weekly. In some embodiments, the amount of limus druguseful for combination with the BCG is about 5 mg to about 500 mg,including for example about 30 mg to about 400 mg, such as about 100 mgto about 200 mg.

In some embodiments, the amount of the mTOR inhibitor (such as limusdrug, e.g., sirolimus) in the nanoparticle composition is between about5 mg to about 500 mg and the amount of the other agent is about 1 mg/kgto about 200 mg/kg (including for example about 1 mg/kg to about 20mg/kg, about 20 mg/kg to about 40 mg/kg, about 40 mg/kg to about 60mg/kg, about 60 mg/kg to about 80 mg/kg, about 80 mg/kg to about 100mg/kg, about 100 mg/kg to about 120 mg/kg, about 120 mg/kg to about 140mg/kg, about 140 mg/kg to about 200 mg/kg). In some embodiments, theamount of 1 mTOR inhibitor (such as limus drug, e.g., sirolimus) in thenanoparticle composition is between about 30 mg to about 400 mg and theamount of the other agent is about 1 mg/kg to about 200 mg/kg (includingfor example about 1 mg/kg to about 20 mg/kg, about 20 mg/kg to about 40mg/kg, about 40 mg/kg to about 60 mg/kg, about 60 mg/kg to about 80mg/kg, about 80 mg/kg to about 100 mg/kg, about 100 mg/kg to about 120mg/kg, about 120 mg/kg to about 140 mg/kg, about 140 mg/kg to about 200mg/kg). In some embodiments, the amount of mTOR inhibitor (such as limusdrug, e.g., sirolimus) in the nanoparticle composition is between about100 mg to about 200 mg and the amount of the other agent is about 1mg/kg to about 200 mg/kg (including for example about 1 mg/kg to about20 mg/kg, about 20 mg/kg to about 40 mg/kg, about 40 mg/kg to about 60mg/kg, about 60 mg/kg to about 80 mg/kg, about 80 mg/kg to about 100mg/kg, about 100 mg/kg to about 120 mg/kg, about 120 mg/kg to about 140mg/kg, about 140 mg/kg to about 200 mg/kg).

In some embodiments, the appropriate doses of other agents areapproximately those already employed in clinical therapies wherein theother agent are administered alone or in combination with other agents.

Nanoparticle Compositions

The nanoparticle compositions described herein comprise nanoparticlescomprising (in various embodiments consisting essentially of) an mTORinhibitor (such as a limus drug, for example sirolimus). Thenanoparticles may further comprise a carrier protein (e.g., an albuminsuch as human serum albumin or human albumin). Nanoparticles of poorlywater soluble drugs have been disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, 6,537,579, 7,820,788, and also in U.S.Pat. Pub. Nos. 2006/0263434, and 2007/0082838; PCT Patent ApplicationWO08/137148, each of which is incorporated by reference in theirentirety.

In some embodiments, the composition comprises nanoparticles with anaverage or mean diameter of no greater than about 1000 nanometers (nm),such as no greater than about (or less than about) any of 900, 800, 700,600, 500, 400, 300, 200, and 100 nm. In some embodiments, the average ormean diameters of the nanoparticles is no greater than about 200 nm(such as no greater than about 150 nm). In some embodiments, the averageor mean diameters of the nanoparticles is no greater than about 150 nm.In some embodiments, the average or mean diameters of the nanoparticlesis no greater than about 100 nm. In some embodiments, the average ormean diameter of the nanoparticles is about 20 nm to about 400 nm. Insome embodiments, the average or mean diameter of the nanoparticles isabout 40 nm to about 200 nm. In some embodiments, the nanoparticles aresterile-filterable.

In some embodiments, the nanoparticles in the composition describedherein have an average diameter of no greater than about 200 nm,including for example no greater than about any one of 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In someembodiments, at least about 50% (for example at least about any one of60%, 70%, 80%, 90%, 95%, or 99%) of the nanoparticles in the compositionhave a diameter of no greater than about 200 nm, including for exampleno greater than about any one of 190, 180, 170, 160, 150, 140, 130, 120,110, 100, 90, 80, 70, or 60 nm. In some embodiments, at least about 50%(for example at least any one of 60%, 70%, 80%, 90%, 95%, or 99%) of thenanoparticles in the composition fall within the range of about 20 nm toabout 400 nm, including for example about 20 nm to about 200 nm, about40 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about150 nm, about 50 nm to about 120 nm, and about 60 nm to about 100 nm.

In some embodiments, the carrier protein (e.g., an albumin) hassulfhydryl groups that can form disulfide bonds. In some embodiments, atleast about 5% (including for example at least about any one of 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of carrier protein(e.g., an albumin) in the nanoparticle portion of the composition arecrosslinked (for example crosslinked through one or more disulfidebonds).

In some embodiments, the nanoparticles comprising the mTOR inhibitor(such as a limus drug, e.g., sirolimus) are coated with a carrierprotein (e.g., an albumin such as human albumin or human serum albumin).In some embodiments, the composition comprises an mTOR inhibitor (suchas a limus drug, for example sirolimus) in both nanoparticle andnon-nanoparticle forms (e.g., in the form of solutions or in the form ofsoluble carrier protein/nanoparticle complexes), wherein at least aboutany one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the mTOR inhibitor(such as a limus drug, e.g., sirolimus) in the composition are innanoparticle form. In some embodiments, the mTOR inhibitor (such as alimus drug, e.g., sirolimus) in the nanoparticles constitutes more thanabout any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thenanoparticles by weight. In some embodiments, the nanoparticles have anon-polymeric matrix. In some embodiments, the nanoparticles comprise acore of an mTOR inhibitor (such as a limus drug, for example sirolimus)that is substantially free of polymeric materials (such as polymericmatrix).

In some embodiments, the composition comprises a carrier protein (e.g.,an albumin) in both nanoparticle and non-nanoparticle portions of thecomposition, wherein at least about any one of 50%, 60%, 70%, 80%, 90%,95%, or 99% of the carrier protein (e.g., an albumin) in the compositionare in non-nanoparticle portion of the composition.

In some embodiments, the weight ratio of an albumin (such as humanalbumin or human serum albumin) and a mTOR inhibitor in the nanoparticlecomposition is about 18:1 or less, such as about 15:1 or less, forexample about 10:1 or less. In some embodiments, the weight ratio of analbumin (such as human albumin or human serum albumin) and an mTORinhibitor (such as a limus drug, for example sirolimus) in thecomposition falls within the range of any one of about 1:1 to about18:1, about 2:1 to about 15:1, about 3:1 to about 13:1, about 4:1 toabout 12:1, about 5:1 to about 10:1. In some embodiments, the weightratio of an albumin and an mTOR inhibitor (such as a limus drug, forexample sirolimus) in the nanoparticle portion of the composition isabout any one of 1:2, 1:3, 1:4, 1:5, 1:9, 1:10, 1:15, or less. In someembodiments, the weight ratio of the albumin (such as human albumin orhuman serum albumin) and the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) in the composition is any one of the following: about 1:1 toabout 18:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1to about 1:1.

In some embodiments, the nanoparticle composition comprises one or moreof the above characteristics.

The nanoparticles described herein may be present in a dry formulation(such as lyophilized composition) or suspended in a biocompatiblemedium. Suitable biocompatible media include, but are not limited to,water, buffered aqueous media, saline, buffered saline, optionallybuffered solutions of amino acids, optionally buffered solutions ofproteins, optionally buffered solutions of sugars, optionally bufferedsolutions of vitamins, optionally buffered solutions of syntheticpolymers, lipid-containing emulsions, and the like.

In some embodiments, the pharmaceutically acceptable carrier comprises acarrier protein (e.g., an albumin such as human albumin or human serumalbumin). Examples of suitable carrier proteins include proteinsnormally found in blood or plasma, which include, but are not limitedto, an albumin, immunoglobulin including IgA, lipoproteins,apolipoprotein B, α-acid glycoprotein, β-2-macroglobulin, thyroglobulin,transferrin, fibronectin, factor VII, factor VIII, factor IX, factor X,and the like. In some embodiments, the carrier protein is non-bloodprotein, such as casein, α-lactalbumin, β-lactoglobulin. The proteinsmay either be natural in origin or synthetically prepared. In someembodiments, the protein is an albumin, such as human albumin or humanserum albumin. In some embodiments, the albumin is a recombinantalbumin.

Human serum albumin (HSA) is a highly soluble globular protein of M_(r)65K and consists of 585 amino acids. HSA is the most abundant protein inthe plasma and accounts for 70-80% of the colloid osmotic pressure ofhuman plasma. The amino acid sequence of HSA contains a total of 17disulfide bridges, one free thiol (Cys 34), and a single tryptophan (Trp214). Intravenous use of HSA solution has been indicated for theprevention and treatment of hypovolumic shock (see, e.g., Tullis, JAMA,237: 355-360, 460-463, (1977)) and Houser et al., Surgery, Gynecologyand Obstetrics, 150: 811-816 (1980)) and in conjunction with exchangetransfusion in the treatment of neonatal hyperbilirubinemia (see, e.g.,Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)).Other albumins are contemplated, such as bovine serum albumin. Use ofsuch non-human albumins could be appropriate, for example, in thecontext of use of these compositions in non-human mammals, such as theveterinary (including domestic pets and agricultural context). Humanserum albumin (HSA) has multiple hydrophobic binding sites (a total ofeight for fatty acids, an endogenous ligand of HSA) and binds a diverseset of drugs, especially neutral and negatively charged hydrophobiccompounds (Goodman et al., The Pharmacological Basis of Therapeutics,9th ed, McGraw-Hill New York (1996)). Two high affinity binding siteshave been proposed in subdomains IIA and IIIA of HSA, which are highlyelongated hydrophobic pockets with charged lysine and arginine residuesnear the surface which function as attachment points for polar ligandfeatures (see, e.g., Fehske et al., Biochem. Pharmcol., 30, 687-92(198a), Vorum, Dan. Med. Bull., 46, 379-99 (1999), Kragh-Hansen, Dan.Med. Bull., 1441, 131-40 (1990), Curry et al., Nat. Struct. Biol., 5,827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46 (1999), He etal., Nature, 358, 209-15 (199b), and Carter et al., Adv. Protein. Chem.,45, 153-203 (1994)). Sirolimus and propofol have been shown to bind HSA(see, e.g., Paal et al., Eur. J. Biochem., 268(7), 2187-91 (200a),Purcell et al., Biochim. Biophys. Acta, 1478(a), 61-8 (2000), Altmayeret al., Arzneimittelforschung, 45, 1053-6 (1995), and Garrido et al.,Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). In addition,docetaxel has been shown to bind to human plasma proteins (see, e.g.,Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

The carrier protein (e.g., an albumin such as human albumin or humanserum albumin) in the composition generally serves as a carrier for themTOR inhibitor, i.e., the albumin in the composition makes the mTORinhibitor (such as a limus drug, e.g., sirolimus) more readilysuspendable in an aqueous medium or helps maintain the suspension ascompared to compositions not comprising a carrier protein. This canavoid the use of toxic solvents (or surfactants) for solubilizing themTOR inhibitor, and thereby can reduce one or more side effects ofadministration of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) into an individual (such as a human). Thus, in someembodiments, the composition described herein is substantially free(such as free) of surfactants, such as Cremophor (or polyoxyethylatedcastor oil, including Cremophor EL® (BASF)). In some embodiments, thenanoparticle composition is substantially free (such as free) ofsurfactants. A composition is “substantially free of Cremophor” or“substantially free of surfactant” if the amount of Cremophor orsurfactant in the composition is not sufficient to cause one or moreside effect(s) in an individual when the nanoparticle composition isadministered to the individual. In some embodiments, the nanoparticlecomposition contains less than about any one of 20%, 15%, 10%, 7.5%, 5%,2.5%, or 1% organic solvent or surfactant. In some embodiments, thecarrier protein is an albumin. In some embodiments, the albumin is humanalbumin or human serum albumin. In some embodiments, the albumin isrecombinant albumin.

The amount of a carrier protein such as an albumin in the compositiondescribed herein will vary depending on other components in thecomposition. In some embodiments, the composition comprises a carrierprotein such as an albumin in an amount that is sufficient to stabilizethe mTOR inhibitor (such as a limus drug, e.g., sirolimus) in an aqueoussuspension, for example, in the form of a stable colloidal suspension(such as a stable suspension of nanoparticles). In some embodiments, thecarrier protein such as an albumin is in an amount that reduces thesedimentation rate of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) in an aqueous medium. For particle-containing compositions,the amount of the carrier protein such as an albumin also depends on thesize and density of nanoparticles of the mTOR inhibitor.

An mTOR inhibitor (such as a limus drug, for example sirolimus) is“stabilized” in an aqueous suspension if it remains suspended in anaqueous medium (such as without visible precipitation or sedimentation)for an extended period of time, such as for at least about any of 0.1,0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60,or 72 hours. The suspension is generally, but not necessarily, suitablefor administration to an individual (such as human). Stability of thesuspension is generally (but not necessarily) evaluated at a storagetemperature (such as room temperature (such as 20-25° C.) orrefrigerated conditions (such as 4° C.)). For example, a suspension isstable at a storage temperature if it exhibits no flocculation orparticle agglomeration visible to the naked eye or when viewed under theoptical microscope at 1000 times, at about fifteen minutes afterpreparation of the suspension. Stability can also be evaluated underaccelerated testing conditions, such as at a temperature that is higherthan about 40° C.

In some embodiments, the carrier protein (e.g., an albumin) is presentin an amount that is sufficient to stabilize the mTOR inhibitor (such asa limus drug, e.g., sirolimus) in an aqueous suspension at a certainconcentration. For example, the concentration of the mTOR inhibitor(such as a limus drug, e.g., sirolimus) in the composition is about 0.1mg/ml to about 100 mg/ml, including for example any of about 0.1 mg/mlto about 50 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 1 mg/ml toabout 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 mg/ml to about 6mg/ml, or about 5 mg/ml. In some embodiments, the concentration of themTOR inhibitor (such as a limus drug, e.g., sirolimus) is at least aboutany of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml. In some embodiments, thecarrier protein (e.g., an albumin) is present in an amount that avoidsuse of surfactants (such as Cremophor), so that the composition is freeor substantially free of surfactant (such as Cremophor).

In some embodiments, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g. about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v),about 40% (w/v), or about 50% (w/v)) of carrier protein (e.g., analbumin). In some embodiments, the composition, in liquid form,comprises about 0.5% to about 5% (w/v) of carrier protein (e.g., analbumin).

In some embodiments, the weight ratio of a carrier protein (e.g., analbumin) to the mTOR inhibitor (such as a limus drug, e.g., sirolimus)in the nanoparticle composition is such that a sufficient amount of mTORinhibitor binds to, or is transported by, the cell. While the weightratio of a carrier protein (e.g., an albumin) to mTOR inhibitor willhave to be optimized for different carrier protein (e.g., an albumin)and mTOR inhibitor combinations, generally the weight ratio of carrierprotein (e.g., an albumin), to mTOR inhibitor (such as a limus drug,e.g., sirolimus) (w/w) is about 0.01:1 to about 100:1, about 0.02:1 toabout 50:1, about 0.05:1 to about 20:1, about 0.1:1 to about 20:1, about1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1,about 4:1 to about 10:1, about 5:1 to about 9:1, or about 9:1. In someembodiments, the carrier protein (e.g., an albumin) to mTOR inhibitorweight ratio is about any of 18:1 or less, 15:1 or less, 14:1 or less,13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 or less, 8:1or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, and 3:1 orless. In some embodiments, the carrier protein is an albumin. In someembodiments, the weight ratio of the albumin (such as human albumin orhuman serum albumin) to the mTOR inhibitor in the composition is any oneof the following: about 1:1 to about 18:1, about 1:1 to about 15:1,about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

In some embodiments, the carrier protein (e.g., an albumin) allows thecomposition to be administered to an individual (such as human) withoutsignificant side effects. In some embodiments, the carrier protein(e.g., an albumin such as human serum albumin or human albumin) is in anamount that is effective to reduce one or more side effects ofadministration of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) to a human. The term “reducing one or more side effects” ofadministration of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) refers to reduction, alleviation, elimination, or avoidanceof one or more undesirable effects caused by the mTOR inhibitor, as wellas side effects caused by delivery vehicles (such as solvents thatrender the limus drugs suitable for injection) used to deliver the mTORinhibitor. Such side effects include, for example, myelosuppression,neurotoxicity, hypersensitivity, inflammation, venous irritation,phlebitis, pain, skin irritation, peripheral neuropathy, neutropenicfever, anaphylactic reaction, venous thrombosis, extravasation, andcombinations thereof. These side effects, however, are merely exemplaryand other side effects, or combination of side effects, associated withlimus drugs (such as sirolimus) can be reduced.

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) and an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 200 nm. In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) and an albumin(such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm.In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) and an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 150 nm (for example about 100 nm). In someembodiments, the nanoparticle compositions described herein comprisesnanoparticles comprising sirolimus and human albumin (such as humanserum albumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm (for example about 100 nm).

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) and an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 200 nm, wherein the weight ratio of the albuminand the mTOR inhibitor in the composition is no greater than about 9:1(such as about 9:1 or about 8:1). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) and an albumin(such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm,wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the nanoparticle compositions describedherein comprises nanoparticles comprising an mTOR inhibitor (such as alimus drug, for example sirolimus) and an albumin (such as human albuminor human serum albumin), wherein the nanoparticles have an averagediameter of about 150 nm, wherein the weight ratio of the albumin andthe mTOR inhibitor in the composition is no greater than about 9:1 (suchas about 9:1 or about 8:1). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprisingsirolimus and human albumin (such as human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm(for example about 100 nm), wherein the weight ratio of albumin andsirolimus inhibitor in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) coated with an albumin (such as humanalbumin or human serum albumin). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) coated with analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) coated with an albumin (such as humanalbumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm. In some embodiments,the nanoparticle compositions described herein comprises nanoparticlescomprising an mTOR inhibitor (such as a limus drug, for examplesirolimus) coated with an albumin (such as human albumin or human serumalbumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm (for example about 100 nm). In someembodiments, the nanoparticle compositions described herein comprisesnanoparticles comprising sirolimus coated with human albumin (such ashuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 150 nm (for example about 100 nm).

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) coated with an albumin (such as humanalbumin or human serum albumin), wherein the weight ratio of the albuminand the mTOR inhibitor in the composition is no greater than about 9:1(such as about 9:1 or about 8:1). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) coated with analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm,wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the nanoparticle compositions describedherein comprises nanoparticles comprising an mTOR inhibitor (such as alimus drug, for example sirolimus) coated with an albumin (such as humanalbumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm, wherein the weightratio of the albumin and the mTOR inhibitor in the composition is nogreater than about 9:1 (such as about 9:1 or about 8:1). In someembodiments, the nanoparticle compositions described herein comprisesnanoparticles comprising an mTOR inhibitor (such as a limus drug, forexample sirolimus) coated with an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof about 150 nm, wherein the weight ratio of the albumin and the mTORinhibitor in the composition is no greater than about 9:1 (such as about9:1 or about 8:1). In some embodiments, the nanoparticle compositionsdescribed herein comprises nanoparticles comprising sirolimus coatedwith human albumin (such as human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm(for example about 100 nm), wherein the weight ratio of albumin and thesirolimus in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) stabilized by an albumin (such as humanalbumin or human serum albumin). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) stabilized by analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) stabilized by an albumin (such as humanalbumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm. In some embodiments,the nanoparticle compositions described herein comprises nanoparticlescomprising an mTOR inhibitor (such as a limus drug, for examplesirolimus) stabilized by an albumin (such as human albumin or humanserum albumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm (for example about 100 nm). In someembodiments, the nanoparticle compositions described herein comprisesnanoparticles comprising sirolimus stabilized by human albumin (such ashuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 150 nm (for example about 100 nm).

In some embodiments, the nanoparticle compositions described hereincomprises nanoparticles comprising an mTOR inhibitor (such as a limusdrug, for example sirolimus) stabilized by an albumin (such as humanalbumin or human serum albumin), wherein the weight ratio of the albuminand the mTOR inhibitor in the composition is no greater than about 9:1(such as about 9:1 or about 8:1). In some embodiments, the nanoparticlecompositions described herein comprises nanoparticles comprising an mTORinhibitor (such as a limus drug, for example sirolimus) stabilized by analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm,wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the nanoparticle compositions describedherein comprises nanoparticles comprising an mTOR inhibitor (such as alimus drug, for example sirolimus) stabilized by an albumin (such ashuman albumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm, wherein the weightratio of the albumin and the mTOR inhibitor in the composition is nogreater than about 9:1 (such as about 9:1 or about 8:1). In someembodiments, the nanoparticle compositions described herein comprisesnanoparticles comprising an mTOR inhibitor (such as a limus drug, forexample sirolimus) stabilized by an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof about 150 nm, wherein the weight ratio of the albumin and the mTORinhibitor in the composition is no greater than about 9:1 (such as about9:1 or about 8:1). In some embodiments, the nanoparticle compositionsdescribed herein comprises nanoparticles comprising sirolimus stabilizedby human albumin (such as human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm(for example about 100 nm), wherein the weight ratio of albumin and thesirolimus in the composition is about 9:1 or about 8:1.

In some embodiments, the nanoparticle composition comprisesNab-sirolimus. In some embodiments, the nanoparticle composition isNab-sirolimus. Nab-sirolimus is a formulation of sirolimus stabilized byhuman albumin USP, which can be dispersed in directly injectablephysiological solution. The weight ratio of human albumin and sirolimusis about 8:1 to about 9:1. When dispersed in a suitable aqueous mediumsuch as 0.9% sodium chloride injection or 5% dextrose injection,Nab-sirolimus forms a stable colloidal suspension of sirolimus. The meanparticle size of the nanoparticles in the colloidal suspension is about100 nanometers. Since HSA is freely soluble in water, Nab-sirolimus canbe reconstituted in a wide range of concentrations ranging from dilute(0.1 mg/ml sirolimus) to concentrated (20 mg/ml sirolimus), includingfor example about 2 mg/ml to about 8 mg/ml, or about 5 mg/ml.

Methods of making nanoparticle compositions are known in the art. Forexample, nanoparticles containing mTOR inhibitor (such as a limus drug,e.g., sirolimus) and carrier protein (e.g., an albumin such as humanserum albumin or human albumin) can be prepared under conditions of highshear forces (e.g., sonication, high pressure homogenization, or thelike). These methods are disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, 6,537,579 and 7,820,788 and also inU.S. Pat. Pub. Nos. 2007/0082838, 2006/0263434 and PCT ApplicationWO08/137148.

Briefly, the mTOR inhibitor (such as a limus drug, e.g., sirolimus) isdissolved in an organic solvent, and the solution can be added to acarrier protein solution such as an albumin solution. The mixture issubjected to high pressure homogenization. The organic solvent can thenbe removed by evaporation. The dispersion obtained can be furtherlyophilized. Suitable organic solvent include, for example, ketones,esters, ethers, chlorinated solvents, and other solvents known in theart. For example, the organic solvent can be methylene chloride orchloroform/ethanol (for example with a ratio of 1:9, 1:8, 1:7, 1:6, 1:5,1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1).

mTOR Inhibitor

The methods described herein in some embodiments comprise administrationof nanoparticle compositions of mTOR inhibitors. “mTOR inhibitor” usedherein refers to an inhibitor of mTOR. mTOR is aserine/threonine-specific protein kinase downstream of thephosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway, anda key regulator of cell survival, proliferation, stress, and metabolism.mTOR pathway dysregulation has been found in many human carcinomas, andmTOR inhibition produced substantial inhibitory effects on tumorprogression.

The mammalian target of rapamycin (mTOR) (also known as mechanistictarget of rapamycin or FK506 binding protein 12-rapamycin associatedprotein 1 (FRAP1)) is an atypical serine/threonine protein kinase thatis present in two distinct complexes, mTOR Complex 1 (mTORC1) and mTORComplex 2 (mTORC2). mTORC1 is composed of mTOR, regulatory-associatedprotein of mTOR (Raptor), mammalian lethal with SEC13 protein 8 (MLST8),PRAS40 and DEPTOR (Kim et al. (2002). Cell 110: 163-75; Fang et al.(2001). Science 294 (5548): 1942-5). mTORC1 integrates four major signalinputs: nutrients (such as amino acids and phosphatidic acid), growthfactors (insulin), energy and stress (such as hypoxia and DNA damage).Amino acid availability is signaled to mTORC1 via a pathway involvingthe Rag and Ragulator (LAMTOR1-3) Growth factors and hormones (e.g.insulin) signal to mTORC1 via Akt, which inactivates TSC2 to preventinhibition of mTORC1. Alternatively, low ATP levels lead to theAMPK-dependent activation of TSC2 and phosphorylation of raptor toreduce mTORC1 signaling proteins.

Active mTORC1 has a number of downstream biological effects includingtranslation of mRNA via the phosphorylation of downstream targets(4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1),ribosome biogenesis, and activation of transcription leading tomitochondrial metabolism or adipogenesis. Accordingly, mTORC1 activitypromotes either cellular growth when conditions are favorable orcatabolic processes during stress or when conditions are unfavorable.

mTORC2 is composed of mTOR, rapamycin-insensitive companion of mTOR(RICTOR), GβL, and mammalian stress-activated protein kinase interactingprotein 1 (mSIN1). In contrast to mTORC1, for which many upstreamsignals and cellular functions have been defined (see above), relativelylittle is known about mTORC2 biology. mTORC2 regulates cytoskeletalorganization through its stimulation of F-actin stress fibers, paxillin,RhoA, Rac1, Cdc42, and protein kinase C α (PKCα). It had been observedthat knocking down mTORC2 components affects actin polymerization andperturbs cell morphology (Jacinto et al. (2004). Nat. Cell Biol. 6,1122-1128; Sarbassov et al. (2004). Curr. Biol. 14, 1296-1302). Thissuggests that mTORC2 controls the actin cytoskeleton by promotingprotein kinase Cα (PKCα) phosphorylation, phosphorylation of paxillinand its relocalization to focal adhesions, and the GTP loading of RhoAand Rac1. The molecular mechanism by which mTORC2 regulates theseprocesses has not been determined.

In some embodiments, the mTOR inhibitor is an inhibitor of mTORC1. Insome embodiments, the mTOR inhibitor is an inhibitor of mTORC2.

In some embodiments, the mTOR inhibitor is a limus drug, which includessirolimus and its analogues. Examples of limus drugs include, but arenot limited to, temsirolimus (CCI-779), everolimus (RAD001),ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578),pimecrolimus, and tacrolimus (FK-506). In some embodiments, the limusdrug is selected from the group consisting of temsirolimus (CCI-779),everolimus (RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669),zotarolimus (ABT-578), pimecrolimus, and tacrolimus (FK-506).

In some embodiments, the mTOR inhibitor is sirolimus. Sirolimus ismacrolide antibiotic that complexes with FKBP-12 and inhibits the mTORpathway by binding mTORC1.

In some embodiments, the mTOR inhibitor is selected from the groupconsisting of sirolimus (rapamycin), BEZ235 (NVP-BEZ235), everolimus(also known as RAD001 and sold under the trademarks Zortress®,Certican®, and Afinitor®), AZD8055, temsirolimus (also known as CCI-779and sold under the trademark Torisel®), PI-103, Ku-0063794, INK 128,AZD2014, NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torin 1,WAY-600, WYE-125132, WYE-687, GSK2126458, PF-05212384 (PKI-587), PP-121,OSI-027, Palomid 529, PP242, XL765, GSK1059615, WYE-354, and eforolimus(also known as ridaforolimus or deforolimus).

BEZ235 (NVP-BEZ235) is an imidazoquilonine derivative that is an mTORC1catalytic inhibitor (Roper J, et al. PLoS One, 2011, 6(9), e25132).Everolimus is the 40-O-(2-hydroxyethyl) derivative of rapamycin andbinds the cyclophilin FKBP-12, and this complex also mTORC1. AZD8055 isa small molecule that inhibits the phosphorylation of mTORC1 (p70S6K and4E-BP1). Temsirolimus is a small molecule that forms a complex with theFK506-binding protein and prohibits the activation of mTOR when itresides in the mTORC1complex. PI-103 is a small molecule that inhibitsthe activation of the rapamycin-sensitive (mTORC1) complex (Knight etal. (2006) Cell. 125: 733-47). KU-0063794 is a small molecule thatinhibits the phosphorylation of mTORC1 at Ser2448 in a dose-dependentand time-dependent manner. INK 128, AZD2014, NVP-BGT226, CH5132799,WYE-687, and are each small molecule inhibitors of mTORC1. PF-04691502inhibits mTORC1 activity. GDC-0980 is an orally bioavailable smallmolecule that inhibits Class I PI3 Kinase and TORC1. Torin 1 is a potentsmall molecule inhibitor of mTOR. WAY-600 is a potent, ATP-competitiveand selective inhibitor of mTOR. WYE-125132 is an ATP-competitive smallmolecule inhibitor of mTORC1. GSK2126458 is an inhibitor of mTORC1.PKI-587 is a highly potent dual inhibitor of PI3Kα, PI3Kγ and mTOR.PP-121 is a multi-target inhibitor of PDGFR, Hck, mTOR, VEGFR2, Src andAbl. OSI-027 is a selective and potent dual inhibitor of mTORC1 andmTORC2 with IC50 of 22 nM and 65 nM, respectively. Palomid 529 is asmall molecule inhibitor of mTORC1 that lacks affinity for ABCB1/ABCG2and has good brain penetration (Lin et al. (2013) Int J Cancer DOI:10.1002/ijc.28126 (e-published ahead of print). PP242 is a selectivemTOR inhibitor. XL765 is a dual inhibitor of mTOR/PI3k for mTOR, p110α,p110β, p110γ and p110δ. GSK1059615 is a novel and dual inhibitor ofPI3Kα, PI3Kβ, PI3Kδ, PI3Kγ and mTOR. WYE-354 inhibits mTORC1 in HEK293cells (0.2 μM-5 μM) and in HUVEC cells (10 nM-1 μM). WYE-354 is apotent, specific and ATP-competitive inhibitor of mTOR. Deforolimus(Ridaforolimus, AP23573, MK-8669) is a selective mTOR inhibitor.

Other Components in the Nanoparticle Compositions

The nanoparticles described herein can be present in a composition thatinclude other agents, excipients, or stabilizers. For example, toincrease stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bile saltsof bile acids consisting of glycocholic acid, cholic acid,chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid,taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid,dehydrocholic acid and others; phospholipids including lecithin (eggyolk) based phospholipids which include the followingphosphatidylcholines: palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearyolphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the nanoparticle composition (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331). The following formulations and methods aremerely exemplary and are in no way limiting. Formulations suitable fororal administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, and (d) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Examples of suitable carriers, excipients, and diluents include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

In some embodiments, the composition is formulated to have a pH range ofabout 4.5 to about 9.0, including for example pH ranges of any of about5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. Insome embodiments, the pH of the composition is formulated to no lessthan about 6, including for example no less than about any of 6.5, 7, or8 (such as about 8). The composition can also be made to be isotonicwith blood by the addition of a suitable tonicity modifier, such asglycerol.

Kits, Medicines, and Compositions

The invention also provides kits, medicines, compositions, and unitdosage forms for use in any of the methods described herein.

Kits of the invention include one or more containers comprising limusdrug-containing nanoparticle compositions (or unit dosage forms and/orarticles of manufacture) and/or another agent (such as the agentsdescribed herein), and in some embodiments, further compriseinstructions for use in accordance with any of the methods describedherein. The kit may further comprise a description of selection anindividual suitable or treatment. Instructions supplied in the kits ofthe invention are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

For example, in some embodiments, the kit comprises a) a compositioncomprising nanoparticles comprising mTOR inhibitor (such as a limusdrug) and an albumin (such as human serum albumin), and b) instructionsfor administering the nanoparticle composition for treatment of bladdercancer. In some embodiments, the kit comprises a) a compositioncomprising nanoparticles comprising mTOR inhibitor (such as a limusdrug) and an albumin (such as human serum albumin), b) an effectiveamount of another agent, wherein the other agent inhibits microtubuledisassembly, and c) instructions for administering (such asadministering intravesicularly or intravenously) the nanoparticlecomposition and the other agents for treatment of bladder cancer. Thenanoparticles and the other agents can be present in separate containersor in a single container. For example, the kit may comprise one distinctcomposition or two or more compositions wherein one compositioncomprises nanoparticles and one composition comprises another agent.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., seled Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation. The present application thus also provides articles ofmanufacture, which include vials (such as sealed vials), bottles, jars,flexible packaging, and the like.

The instructions relating to the use of the nanoparticle compositionsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Forexample, kits may be provided that contain sufficient dosages of themTOR inhibitor (such as a limus drug, e.g., sirolimus) as disclosedherein to provide effective treatment of an individual for an extendedperiod, such as any of a week, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4months, 5 months, 7 months, 8 months, 9 months, or more. Kits may alsoinclude multiple unit doses of the mTOR inhibitor (such as a limus drug)and pharmaceutical compositions and instructions for use and packaged inquantities sufficient for storage and use in pharmacies, for example,hospital pharmacies and compounding pharmacies.

Also provided are medicines, compositions, and unit dosage forms usefulfor the methods described herein. In some embodiments, there is provideda medicine (or composition) for use in treating bladder cancer,comprising nanoparticles comprising an mTOR inhibitor (such as a limusdrug) and an albumin (such as human serum albumin). In some embodiments,there is provided a medicine (or composition or a unit dosage form) foruse in treating bladder cancer in conjunction with another agent,comprising nanoparticles comprising a limus drug and an albumin (such ashuman serum albumin), wherein the other agent inhibits microtubuledisassembly. In some embodiments, there is provided a medicine (orcomposition or a unit dosage form) for use in treating bladder cancer,comprising nanoparticles comprising a limus drug and an albumin (such ashuman serum albumin) and another agent, wherein the other agent inhibitsmicrotubule disassembly.

Exemplary Embodiments

The present application in some embodiments provides a method oftreating bladder cancer in an individual, comprising administering tothe individual an effective amount of a composition comprisingnanoparticles comprising a limus drug and an albumin.

In some embodiments according to (e.g., as applied to) the method above,the nanoparticle composition is administered intravesicularly.

In some embodiments according to (e.g., as applied to) any one of themethods above, the bladder cancer is non-muscle-invasive bladder cancer.

In some embodiments according to (e.g., as applied to) any one of themethods above, the bladder cancer is refractory to treatment with BCG,mitomycin C, or interferon.

In some embodiments according to (e.g., as applied to) any one of themethods above, the nanoparticle composition is administered at leastonce weekly.

In some embodiments according to (e.g., as applied to) any one of themethods above, the dose of limus drug in the nanoparticle composition isabout 5 mg to about 500 mg (such as about 30 mg to about 400 mg.

In some embodiments according to (e.g., as applied to) any one of themethods above, the nanoparticle composition is administered at a volumeof about 20 ml to about 150 ml.

In some embodiments according to (e.g., as applied to) any one of themethods above, the nanoparticle composition is administeredintravesicularly, and wherein the composition is retained in the bladderfor about 30 minutes to about 4 hours.

In some embodiments according to (e.g., as applied to) any one of themethods above, further comprising administering an effective amount of asecond therapeutic agent.

In some embodiments according to (e.g., as applied to) any one of themethods above, the second therapeutic agent is an immunotherapeuticagent, such as BCG. In some embodiments, the BCG is administeredintravesicularly, e.g., at the dose of about 8 mg to about 100 mg.

In some embodiments according to (e.g., as applied to) any one of themethods above, the method further comprises administering to theindividual a therapeutic agent. In some embodiments, the therapeuticagent is selected from the group consisting of an alkylating agent, ananthracycline antibiotic, an antimetabolite, an indolequinone, a taxane,and a platinum-based agent. In some embodiments, the therapeutic agentis selected from the group consisting of mitomycin, epirubicin,doxorubicin, valrubicin, gemcitabine, apaziquone, docetaxel, paclitaxel,and cisplatin.

In some embodiments according to (e.g., as applied to) any one of themethods above, the limus drug is sirolimus.

In some embodiments according to (e.g., as applied to) any one of themethods above, the nanoparticles in the composition have an averagediameter of no greater than about 200 nm.

In some embodiments according to (e.g., as applied to) any one of themethods above, the limus drug in the nanoparticles are coated withalbumin.

In some embodiments according to (e.g., as applied to) any one of themethods above, the bladder cancer is urothelial carcinoma.

In some embodiments according to (e.g., as applied to) any one of themethods above, the bladder cancer is a high grade bladder cancer.

In some embodiments according to (e.g., as applied to) any one of themethods above, the individual is human.

In some embodiments according to (e.g., as applied to) any one of themethods above, the individual is selected for treatment based on thelevel of one of more of: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin,Tau, SPARC, p73, c-myc, and cyclin D1.

In some embodiments according to (e.g., as applied to) any one of themethods above, further comprising determining the level of one of moreof: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73,c-myc, and cyclin D1 prior to treatment. In some embodiments, the methodfurther comprises selecting the individual for treatment based on a highlevel of one or more of: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin,Tau, SPARC, p73, c-myc, and cyclin D1.

In some embodiments according to (e.g., as applied to) any one of themethods above, further comprising determining the level of one of moreof: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p73,c-myc, and cyclin D1 after the treatment.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

EXAMPLES Example 1 Phase 1 Clinical Trial for Establishing the MaximumDelivered Dose (MDD) and Safety of Nab-Sirolimus (mTOR Inhibitor) forIntravesicular Treatment of BCG-Refractory Non-Muscle Invasive BladderCancer (NMIBC)

Patients with BCG-refractory NMIBC receive Nab-sirolimusintravesicularly by sterile urethral catheterization following resectionof visible tumors during cystoscopy. This study enrolls 15 patients, 3per cohort: 100 mg/week, 100 mg 2×/week (total weekly dose 200 mg), 300mg/week, 200 mg 2×/week (total weekly dose 400 mg), and 400 mg/week for6 weeks of treatment. For each treatment, nab-sirolimus is reconstitutedwith 100 ml 0.9% sodium chloride. Patients are instructed to keep thedrug in the bladder for 2 hours before voiding. If a National CancerInstitute Common Toxicity Criteria (NCI CTC) v3.0 grade 2 local toxicitydevelops, treatment is delayed for 1 dose and resume if the toxicityresolves to grade 1 or less. A DLT (dose limiting toxicity) isconsidered any grade 3 or 4 event, and the patient is immediatelyremoved from the trial. Dose escalation follows the 3+3 rule toestablish the MDD. Six weeks after the last treatment, patients undergoa cystoscopy and biopsy. A complete response (CR) is defined as acancer-negative biopsy at the 6-week post-treatment.

If a patient has a CR, the patient receives additional monthlymaintenance instillations at the maximum dose that particular patientreceived. Cystoscopic examinations are every 3 months, and the patientwill receive therapy until disease progression for a maximum of 6additional instillations.

Systemic and local bladder toxicities are monitored throughout treatmentand maintenance therapy.

Collection and Processing of Samples: Physical exams and collection ofurine and blood samples are performed at enrollment, treatment days, endof treatment, and 6-week follow up. Biopsies of tumor and normal bladdertissue are taken pretreatment, once during treatment prior to day 14dosing, and at the 6-week post-treatment cystoscopy.

Analysis: At each visit, patients are monitored for local bladdertoxicity as defined by the NCI CTC v3.0. At every visit, patients'vitals (weight, blood pressure, and pulse) and updated medical historyare obtained. Urine samples are checked by dipstick for pH and sent tothe laboratory for analysis. Four hours after the nab-sirolimustreatment, blood samples are taken for analysis of serum sirolimuslevels. Complete blood count, basic metabolic panel, hepatic functionalpanel, lipid panel, and coagulation profile are checked for signs ofsystemic toxicity.

Example 2 Efficacy of the Combination of Intravesicular Nab-Sirolimusand BCG in a Genetically Engineered Mouse Model of Bladder Cancer

This preclinical study uses an animal model of progressive bladdercancer. These genetically engineered mice have a targeted deletion ofp53 and PTEN in the bladder epithelium. This combined deletion of p53and PTEN in the bladder epithelium after the delivery of Adeno-Creresults in CIS development at ˜6 weeks after injection, resembling thehuman disease. Thus, we initiate treatment at 6 weeks after Adeno-Creinjection. We use only female mice in our study as an angiocatheter canbe easily passed through the urethra compared to male mice whoseurethras are more convoluted.

Surgery: After adequate sedation is achieved with inhalationalanesthesia, a lubricated angiocatheter (24G) is passed through theurethra. The bladder is irrigated with sterile PBS to ensure return ofurine and thus, proper positioning in the bladder. Urine will be removedand discarded. Treatment will be delivered via the angiocatheter andinto the bladder. Silk suture (5-0) is secured around the urethra toprevent intravesicular treatment from being expelled. Suture remains inplace be until a treatment time of 2 hours is achieved.

Doses: A preliminary in vitro study determines if Nab-sirolimus has anyeffect on BCG viability. A pilot study is performed to determinetolerable dosage of single agents and combination therapy. We start witha BCG dose of 2×10⁶ CFU/ml based on the clinically used dose (1-8×10⁶CFU/ml), and the dose used in a previous preclinical study withBCG-based combination therapy. For Nab-sirolimus, we start with aninitial intravesicular dose of 15 mg/kg. After the conclusion of thepilot study, we establish the specific study dose in mice (n=15/group).Treatment duration is 6 weeks. The single agent cohort undergoes onceweekly treatments of either BCG or nab-sirolimus, with a total indwelltime of 2 hours. The combination cohort receives BCG on Monday, thenNab-sirolimus on Thursday. A control group receives vehicle once weekly.Biweekly weights are utilized to assess drug toxicity. Mice with bodyweight loss of 10-15% or sick appearance is euthanized for humanereasons. As an endpoint, if the tumor is greater than 1.5 cm, the miceis euthanized.

Collection and Processing of Samples: Periodic ultrasonography will beperformed prior to treatment and every 2 weeks thereafter to monitorpresence of tumor and tumor size. Throughout the course of thepreclinical trial, we observe the mice for any physical signs ofdistress or loss of body weight (10-15%), which could indicate toxicityor infection.

Analysis: All surviving mice are sacrificed after 6 weeks ofintravesicular treatment for evaluation of bladder size, weight, andimmunohistochemistry (IHC) analysis. IHC analysis includes hematoxylinand eosin (H&E) staining, markers of the mTOR pathway (pS6, p-AKT,p-4EBP1), and markers of immune cells (to identify macrophages,dendritic cells, T-cells, etc).

Example 3 Evaluation of the Systemic and Target Tissue Drug Exposure ofIntravesicular Nab-Sirolimus in Patients with NMIBC

During phase ½ clinical trials, blood samples are taken 4 hours afternab-sirolimus administration, collected in EDTA-treated tubes, andstored at −80° C. until analysis. The bladder tissue biopsy samples formeasuring sirolimus levels (5 samples per patient) are taken viacystoscopy just prior to dosing on day 14. The tissue samples arecollected into 15 ml cryovials, flash frozen on dry ice, and stored at−80° C. until analysis.

Analysis: Sirolimus levels in serum and in bladder tissue samples aremeasured by liquid chromatography/mass spectrometry (LC/MS). Sirolimusconcentrations in the target bladder tissue are correlated with clinicalefficacy data, and pharmacodynamic biomarkers to help establisheffective biological dose.

Example 4 Evaluation of Target Biomarker Suppression and Other RelevantMolecular Markers in Patient Tissue Samples

We evaluate the activated mTOR levels at baseline and at 6-weekpost-treatment, which help to determine the clinical value forpretreatment screening of patient population. Demonstration of mTORpathway suppression following treatment can provide clear indication ofthe specificity and efficacy of nab-sirolimus and potentially predictclinical response. Several biomarkers are particular relevant to themTOR pathway (p-S6K, p-AKT, p-4EBP1), and bladder cancer (Ki67) and willbe examined. P-S6 is a biomarker of mTOR pathway activation, which isexpected to be reduced when mTOR activity is inhibited. Ki67 is acellular marker for proliferation and is elevated in bladder cancer.Following inhibition of mTORC1 by sirolimus and its analogues, the p-Aktis increased through feedback activation with mTORC2, which may resultin side effects and resistance of sirolimus treatment. Other potentialmolecular markers would include p53, p63, Stathmin, Tau, Ki67, andSPARC.

Collection and Processing of Samples: Prior to treatment, tumor andnormal tissues samples are collected by resection of visible tumorsduring cystoscopy. Additional normal tissue samples (and tumor samplesif available) are collected prior to the 14-day treatment and at 6-weekspost-treatment cystoscopy. The samples are immediately frozen andsectioned.

Analysis: The samples are analyzed by immunohistochemistry (H&Estaining, and with antibodies against p-S6K, p-AKT, p-4EBP1, etc). Theslides are scored by a pathologist for staining intensity of differentbiomarkers. Quantification of proliferating cells are done as describedpreviously. The biomarker status at baseline and during treatment arecorrelated with clinical responses with Fisher's exact test toinvestigate the predictive value of these biomarkers for NMIBC patientstreated with intravesicular Nab-sirolimus therapy.

Example 5 Phase 2 Clinical Study to Evaluate the Efficacy, Safety, andPotential Predictive Biomarkers of Intravesicular Nab-Sirolimus inBCG-Refractory NMIBC

Research Design: The clinical phase 1 dose escalation study is expandedinto this clinical phase 2 study with up to 29 patients enrolled at theMDD to evaluate the utility of nab-sirolimus in the treatment ofBCG-refractory NMIBC as measured by rate of complete responders. Weenroll 10 patients in the first stage (Simon 2-stage method). If 2 ormore patients respond, we enroll an additional 19 in the second stage.If only 1 or no response is observed in the first stage, we terminatethe study for lack of efficacy. Based on our operating characteristic of5% type I error and 20% type II error, the number of patients that isexpected to be enrolled will be 15 on average with a maximum total of 29in order to sufficiently power the study. Patient treatment protocol isthe same as the phase 1 study. If a patient has a CR, the patientreceive additional monthly maintenance instillations at the MDD.Cystoscopic examinations will be performed every 3 months, and thepatient continue therapy until disease progression or for a maximum 6additional instillations. Patients are monitored for local and systemictoxicities throughout the study and maintenance therapy.

Collection and Processing of Samples: Patient tumor and normal bladdertissue samples are taken prior to, during, and after treatment. Bloodand urine samples will also be taken at multiple time points asdescribed preciously.

Analysis: Patient samples are evaluated following procedures describedabove. A CR is defined as a cancer-negative biopsy at the 6-weekpost-treatment cystoscopy. Safety assessment is per standard NCIcriteria.

Example 6 Phase 2 Clinical Study to Evaluate the Efficacy, Safety, andPotential Predictive Biomarkers of Intravesicular Nab-Sirolimus Plus BCGin BCG-Refractory NMIBC

Research Design: BCG is dosed at the clinically used weeklyintravesicular dose of 81 mg BCG in 50 ml saline. BCG can also be usedat half or one third of the standard 81 mg dose. With the establishedMDD of Nab-sirolimus, up to 20 patients are treated with a combinationof intravesicular nab-sirolimus plus BCG. Patient enrollment andstatistical considerations are similar to the study above, with 2 stagesof enrollment. Scheduling of the combination treatment (drugs giventogether or sequentially) are determined from Example 2.

Collection and Processing of Samples: Patient tumor and bladder tissuesamples are taken prior to, during, and after treatment. Blood and urinesamples (for cytokine levels as indicators of local immune response) arealso taken at multiple time points.

Analysis: Patient samples are evaluated following procedures listedabove. Patients are evaluated for efficacy by cystoscopy and biopsy 6weeks post-treatment. A CR is defined as a cancer-negative biopsy at the6-week post-treatment cystoscopy.

Example 7 Phase 2 Clinical Study to Evaluate the Efficacy and Safety ofIntravesicular Nab-Sirolimus and Mitomycin C Combination inBCG-Refractory NMIBC

Design: Appropriate dose of nab-sirolimus is reconstituted in 80-100 mLsaline and administered through intravesicular catheters for a weekly1-2 hour treatment of 6 weeks. Mitomycin C is dosed intravesicularly at40 mg in 40 mL of sterile water for injection, once weekly for 6 weeks.Up to 20 patients are treated with a combination of intravesicularnab-sirolimus and mitomycin C. Scheduling of the combination treatmentis determined from outcomes of Aims 1 and 3. The drugs are givensequentially on the same day, each with 2 hours retention in thebladder. If a patient has a CR, the patient receives additional monthlymaintenance instillations of Nab-sirolimus and mitomycin C. Cystoscopicexaminations are performed every 3 months, and the patient continuetherapy until disease progression or for a maximum 6 additionalinstillations. Patients are monitored for local and systemic toxicitiesthroughout the study and maintenance therapy

Collection and Processing of Samples: Physical exams and collection ofurine and blood samples are performed at enrollment, treatment days, endof treatment, and 6-week follow up. Biopsies of tumor and normal bladdertissue will be taken by cystoscopy pretreatment, once during treatmentprior to day 14 dosing, and at the 6-week post-treatment.

Analysis: Patient samples are evaluated following procedures above.Patients will be evaluated for efficacy by cystoscopy and biopsy 6 weekspost-treatment.

Example 8 Efficacy of the Combination of Nab-Sirolimus and VariousAgents in a Xenograft Model (T24) of Bladder Cancer

T24 human bladder cancer cells are cultured at 37° C. and 5% CO2 in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mLstreptomycin, 800 mg/L NaHCO3 and 3.6 g HEPES. Male NCr nu/nu nude micewill be used. The study will require 100 mice, 5 to 6 weeks old. Animalweight will be 20-30 grams on the day of implantation. Animals areidentified by cage number and ear punch. Approximately one hundred miceare used for the study once the average tumor volumes reachapproximately 100 mm³. The mice will be randomly assigned to 10 studygroups with 10 animals in each group prior to dosing. The study groupsand treatment schedules are listed in the Table below.

Group Treatment (N = 10) Dosing Schedule A Saline weekly for 3 weeks, IVB nab-sirolimus (nab-S) 40 mg/kg, weekly for 3 weeks, IV C Mitomycin C(MMC) 1 mg/kg, weekly for 3 weeks, IP D Cisplatin (Cis) 3 mg/kg, weeklyfor 3 weeks, IP E Gemcitabine (Gem) 50 mg/kg, weekly for 3 weeks, IP FValrubicin (Val) 30 mg/kg, weekly for 3 weeks, IP G MMC + nab-SMitomycin C administered immediately prior to nab-S H Cis + nab-SCisplatin administered immediately prior to nab-S I Gem + nab-SGemcitabine administered immediately prior to nab-S J Val + nab-SValrubicin administered immediately prior to nab-S

To compare the antitumor activity of the combination treatment versussingle agents, 10 treatment groups with mice bearing T24 human bladdercancer xenografts are used, including saline control, nab-sirolimus,mitomycin C, cisplatin, gemcitabine, valrubicin, and the combinationtreatment groups of mitomycin C, cisplatin, gemcitabine, or valrubicineach in combination with nab-sirolimus. The mice are treated for 4weeks, and the tumors are measured with a digital caliper twice weeklyuntil the end of study. Animal body weights are measured twice weekly,and animals are monitored for any physical signs of distress orsignificant loss of body weight (10-15%).

Analysis: Tumor size data are analyzed for tumor growth inhibition byeach treatment regimen. Statistical analysis of tumor growth curves areperformed using ANOVA.

Example 9 Phase 2 Clinical Study to Evaluate the Efficacy and Safety ofIntravesicular Nab-Sirolimus and Gemcitabine Combination inBCG-Refractory NMIBC

Design: Appropriate dose of nab-sirolimus is reconstituted in 100 mLsaline and administered through intravesicular catheters for a treatmentof 6 weeks. Gemcitabine is dosed intravesically at a dose of 2 g inapprox 50 ml once weekly for 6 weeks for 1-2 hours. Up to 20 patientsare treated with a combination of intravesicular nab-sirolimus andmitomycin C. The drugs are given sequentially on the same day, each with2 hours retention in the bladder. If a patient has a CR, the patientreceives additional monthly maintenance instillations of Nab-sirolimusand mitomycin C. Cystoscopic examinations are performed every 3 months,and the patient continues therapy until disease progression or for amaximum 6 additional instillations. Patients are monitored for local andsystemic toxicities throughout the study and maintenance therapy

Collection and Processing of Samples: Physical exams and collection ofurine and blood samples are performed at enrollment, treatment days, endof treatment, and 6-week follow up. Biopsies of tumor and normal bladdertissue will be taken by cystoscopy pretreatment, once during treatmentprior to day 14 dosing, and at the 6-week post-treatment.

Analysis: Patient samples are evaluated following procedures above.Patients will be evaluated for efficacy by cystoscopy and biopsy 6 weekspost-treatment.

1-26. (canceled)
 27. A method of treating non-muscle invasive bladdercancer (NMIBC) in an individual, comprising administering to theindividual an effective amount of nanoparticles comprising a limus drugand an albumin.
 28. The method of claim 27, wherein the NMIBC isrefractory to treatment with BCG, mitomycin C, or interferon.
 29. Themethod of claim 27, wherein the nanoparticle composition is administeredintravesicularly.
 30. The method of claim 27, wherein the nanoparticlecomposition is administered at a frequency of at least once weekly. 31.The method of claim 27, wherein the dose of limus drug in thenanoparticle composition is about 5 to about 500 mg.
 32. The method ofclaim 31, wherein the dose of limus drug in the nanoparticle compositionis about 30 to about 400 mg.
 33. The method of claim 31, wherein thenanoparticle composition is administered at a volume of about 20 toabout 150 ml.
 34. The method of claim 31, wherein the nanoparticlecomposition is administered intravesicularly, and wherein thecomposition is retained in the bladder for about 30 minutes to about 4hours.
 35. The method of claim 27, wherein the composition isadministered with an effective amount of a second therapeutic agent. 36.The method of claim 35, the second therapeutic agent is animmunotherapeutic agent.
 37. The method of claim 35, wherein the secondtherapeutic agent is selected from the group consisting of an alkylatingagent, an anthracycline antibiotic, an antimetabolite, an indolequinone,a taxane, and a platinum-based agent.
 38. The method of claim 37,wherein the second therapeutic agent is selected from the groupconsisting of mitomycin, epirubicin, doxorubicin, valrubicin,gemcitabine, apaziquone, docetaxel, paclitaxel, and cisplatin.
 39. Themethod of claim 27, wherein the limus drug is sirolimus.
 40. The methodof claim 39, wherein the nanoparticles in the composition have anaverage diameter of no greater than about 200 nm.
 41. The method ofclaim 39, wherein the limus drug in the nanoparticles is coated withalbumin.
 42. The method of claim 27, wherein the bladder cancer is ahigh grade bladder cancer.
 43. The method of claim 27, wherein theindividual is human.
 44. The method of claim 27, wherein the individualis selected for treatment based on the level of one of more of: p-S6K,pAKT, p-4EBP1, Ki67, p53, p63, Stathmin, Tau, SPARC, p′73, c-myc, andcyclin D1.
 45. The method of claim 44, further comprising determiningthe level of one of more of: p-S6K, pAKT, p-4EBP1, Ki67, p53, p63,Stathmin, Tau, SPARC, p73, c-myc, and cyclin D1 prior to treatment. 46.The method of claim 44, further comprising selecting the individual fortreatment based on a high level of one or more of: p-S6K, pAKT, p-4EBP1,Ki67, p53, p63, Stathmin, Tau, SPARC, p′73, c-myc, and cyclin D1.